Reaction mass of 2-methylbutyl acetate and pentyl acetate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

OECD 422 and 443: no effects up to the highest dose tested. NOAEL = 1000mg/kg (BASF, 2020 and BASF, 2020)

Link to relevant study records

Reference

Reference 1

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jun 2019 - Jul 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Justification for study design:

The study design was based on ECHA decision no. CCH-D-2114440489-41-01/F:
"Extended one-generation reproductive toxicity study (Annex X, Section 8.7.3.; test method: EU B.56./OECD TG 443) in rats, oral route with the registered substance specified as follows:
- Ten weeks premating exposure duration for the parental (P0) generation;
- Dose level setting shall aim to induce some toxicity at the highest dose level;
- Cohort 1A (Reproductive toxicity);
- Cohort 1B (Reproductive toxicity) without extension to mate the Cohort 1B animals to produce the F2 generation"

The doses were seleceted based on results of a preliminary OECD 422 guideline study with wistar rats (BASF, 2020). No adverse effects were observed up to the limit dose (1000 mg/kg bw/d).

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat is the preferred animal species for reproduction studies according to test guidelines. This strain was selected since extensive historical control data were available for Wistar rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female Wistar rats, strain Crl:WI(Han), supplied by Charles River Laboratories, Research Models and Services, Germany GmbH, which were free from any clinical signs of disease, were used for the investigations. The females were nulliparous and non-pregnant at the beginning of the study. According to a written statement from the breeder, male and female animals were derived from different litters. This was necessary to rule out the possibility of sibling mating.
These animals were used as F0 generation parental animals. All other animals used in this study (F1 generation pups and F1 rearing animals) were derived from the supplier-provided animals.

TEST ANIMALS
- Source:
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (P) 4 wks
- Fasting period before study: no
- Housing:
During the study period, the rats were housed together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp) with the following exceptions:
-From delivery to randomization, during overnight matings (male and female mating partners were housed together), gestation, lactation and females after weaning the animals were housed individually in Polycarbonate cages type III.
-Dams and their litters were housed together until PND 21 in Polycarbonate cages type III.

- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
The animals were housed in fully air-conditioned rooms in which central air conditioning guaranteed a range of temperature of 20-24C and a range of relative humidity of 30-70%. The air change rate was 15 times per hour. There were no or only minimal deviations from these limits.
The day/night cycle was generally 12 hours (12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h).

IN-LIFE DATES: From: To: 12 Jun 2019 to 19 Dec 2019

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% CMC suspension in deionized water with Cremophor EL [10 mg/100 mL]

Details on exposure:

TEST SUBSTANCE PREPARATION
The test substance preparations were prepared in intervals, which took into account the analytical results of the stability verification.
For the preparation of the administration suspensions the test substance was weighed in a graduated flask depending on the dose group, topped up with cooled 0.5% CMC suspension in deionized water with Cremophor EL (10 mg/100mL) and intensely mixed with a magnetic stirrer until it was completely homogenized.
The portions of the test substance preparations for the daily administraion were stored in a refrigerator. Before administraion the preparations were stirred with a magnetic stirrer till room temperature was reached.
Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
According to preliminary test substance preparation analyses, the test substance is not homogenously soluble in water, therefore 0.5% CMC suspension in deionized water with Cremophor EL (10 mg/100mL) was chosen as solubilising agent

Details on mating procedure:

In general, each of the male and female animals was mated overnight at a 1 : 1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group.

The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 6.30 - 9.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted " gestation day (GD) 0" and the following day "gestation day (GD) 1".

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance in 0.5% CMC suspension in deionized water with Cremophor EL (10 mg/100 mL) at room temperature over a period of 7 days (and in a refrigerator over a period of 3 days) had been verified prior to the start of the study.
Samples of the test substance preparations were sent to the analytical laboratory during the study period (at the beginning, towards the middle and towards the end) for verification of the concentrations.
The samples, which were taken for the concentration control analyses at the beginning were also used to verify the homogeneity for the samples of the low and the high concentrations (100 and 1000 mg/kg bw/d). Three samples (one from the top, middle and bottom in each case) were taken for each of these concentrations from the beaker with a magnetic stirrer running.
All test samples, plus a duplicate set of reserve samples, were withdrawn by staff of the Reproduction Toxicology.
The samples towards the middle of the study were analyzed because imprecision occurs during the analysis of the samples from the beginning and lactation of the study.
The samples within this study were labeled with serial numbers. The same number was not used for several samplings. Reserve samples were labeled with 1R, 2R, etc.
The reserve samples were stored at the Laboratory Reproduction Toxicology frozen (at -20 °C) Analysis of these samples were performed in case of equivocal analytical results with the original samples or after loss of/damage to original samples after agreement by the Study Director.

Results
Stability analyses
The stability of test substance in 0.5% CMC suspension in deionized water with Cremophor EL (10 mg/100mL) was demonstrated for a period of 7 days at room temperature and in refrigerator.

Homogeneity analyses
The homogeneous distribution of the test substance in 0.5% CMC suspension in deionized water with Cremophor EL (10 mg/100mL) was demonstrated.

Concentration control analyses
Almost all measured values for Reaction mass of 2-methylbutyl acetate and pentyl acetate were in the expected range of the target concentrations (90 - 110%), demonstrating the correctness of the preparations. Homogeneity analysis demonstrated the homogeneous distribution of the test item in the vehicle.

Two individual values (sample 06 and sample 17) of Reaction mass of 2-methylbutyl acetate and pentyl acetate in 0.5 % sodium carboxymethylcellulose in deionized water with cremophor EL (10 mg/100 mL) were just outside the range of 90 % – 110 % of the nominal concentrations. However, as these were single, minor departures from the tolerance range, the overall concordance of measured and target concentrations of the test item in the dosing preparations was considered to be acceptable.

Frequency of treatment:

once daily

Details on study schedule:

The male and female rats were about 4 weeks old when they arrived from the breeder. During an acclimatization period of about 5 days, animals with lowest and highest body weights were eliminated from the study and used for other purposes. The 100 male and 100 female animals required for the study were about 5 weeks old at the beginning of treatment and their weight variation did not exceed 20 percent of the mean weight of each sex.
The assignment of the animals to the different test groups was carried out using a randomization program, according to their weight three days before the beginning of the administration period (day -3).
After the acclimatization period, the test substance was administered to the animals orally by gavage, once daily at approximately the same time in the morning. Females in labor were not treated. The treatment lasted up to one day prior to sacrifice. The animals of the control group were treated with the vehicle (0.5% CMC suspension in deionized water with Cremophor EL [10 mg/100 mL]), in the same way. The volume administered each day was 10 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.
After a minimum of 10 weeks after the beginning of treatment, males and females from the same dose group were mated, overnight at a ratio of 1 : 1 (for details see: Pairing of F0 generation parental animals).
The females were allowed to deliver and rear their pups (F1 generation pups) until PND 4 (standardization) or PND 21 or 22 (depending on the cohort). Pups of the F1 litter were selected (F1 rearing animals) and assigned to 2 different cohorts which were subjected to specific postweaning examinations.
On PND 4 blood samples were collected from 10 surplus (culled) F1 pups per sex and group. On PND 22 blood samples were collected from 10 surplus F1 pups per sex and group.
Blood samples were taken from 10 animals per test group of the F0 parental animals and cohort 1A animals.
Before weaning of the F1 pups the F0 generation parental male animals were sacrificed. After weaning of F1 pups the F0 generation parental female animals were sacrificed.

F1 rearing animals
Before weaning of the F1 generation pups on PND 21, 45 male and 45 females per group were randomly selected, to be placed into cohorts. Obvious runts (those pups whose body weight was ¾ 25% below the mean body weight of the control group, separate for sexes) were not included.

Cohorts:
Cohort 1A: One male and one female/litter (20/sex/group)
Cohort 1B: One male and one female/litter (25/sex/group)
Selected F1 offspring received the test substance daily by gavage until one day before sacrifice.

Dose / conc.:

100 mg/kg bw/day

Dose / conc.:

300 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

Mortality
A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied. The examinations of these animals were carried out according to the methods established at the pathology laboratory.

Clinical observations
A cageside examination was conducted at least daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and within 5 hours after the administration. Abnormalities and changes were documented daily for each animal. Individual data of daily observations can be found in the raw data.
The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. disability to deliver) were documented on an individual dam basis.
On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.
The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

Food consumption
Generally, food consumption was determined once a week (over a period of 7 days) for male and female F0 parental animals and F1 rearing animals, with the following exceptions:
• Food consumption was not determined after the 10th premating week (male F0 animals) and during the mating period (male and female F0 animals).
• During pregnancy, food consumption of the F0 females with evidence of sperm was determined weekly for GD 0-7, 7-14 and 14-20.
• During lactation, food consumption of the F0 females, which gave birth to a litter was determined for PND 1-4, 4-7, 7-10, 10-14, 14-18 and 18-21.
Food consumption was not determined in the females without positive evidence of sperm during mating and gestation periods, in the females without litter during lactation period and in the females after weaning.

Body weight data
In general, the body weight of the male and female F0 parental animals and F1 rearing animals was determined once a week at the same time of the day (in the morning). The body weight of the F1 rearing animals was determined on the first day of test substance administration and then once a week at the same time of the day (in the morning), with the following exceptions:
• During the mating period of the F0 parental animals, the females were weighed on the day of positive evidence of sperm GD 0 and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition PND 0 and on PND 1, 4, 7, 10, 14, 18 and 21.

The body weight change of the animals was calculated from these results.
Females without positive evidence of sperm, females without litter and females after weaning (PND 21/22), were weighed once a week together with the males. These body weight data were solely used for the calculations of the dose volume; therefore these values are not reported in the Summary.

Detailed clinical observations (DCO)
Detailed clinical observations were performed in all F0 parental animals once before the administration and supsequently once per week and in cohorts 1A and 1B at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of severity, if applicable.
For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 × 37.5 × 25 cm). The following parameters listed were assessed:
1. Abnormal behavior in handling
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Gait abnormalities
12. Lacrimation
13. Palpebral closure
14. Exophthalmos
15. Assessment of the feces excreted during the examination (appearance/consistency)
16. Assessment of the urine excreted during the examination
17. Pupil size

Oestrous cyclicity (parental animals):

Estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats for a minimum of 3 weeks prior to mating. Determination was continued throughout the pairing period until the female exhibited evidence of copulation.
In all cohort 1A females, vaginal smears were collected after vaginal opening until the first cornified smear (estrous) was recorded. The estrous cycle also was evaluated in cohort 1A and 1B females for 2 weeks around PND 75.
At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female and cohort 1A and 1B female with scheduled sacrifice.

Sperm parameters (parental animals):

After the organ weight determination, the following parameters were determined in the right testis or right epididymis of all male F0 parental animals and all cohort 1A males sacrificed on schedule:

Sperm parameter investigations were carried out in a randomized sequence. Parameters and methods:

Parameter Unit Method
Sperm motility % microscopic evaluation
Sperm morphology % vital staining with eosin; microscopic evaluation
Sperm head count Mio/g microscopic evaluation
(cauda epididymis) cauda epididymis with MAKLER chamber after homogenization
Sperm head count Mio/g microscopic evaluation
(testis) testis with MAKLER chamber after homogenization

Sperm morphology and sperm head count (cauda epididymis and testis) were evaluated for the control groups (00/10) and highest dose groups (03/13), only. All morphology slides were archived.

Litter observations:

Litter data

Pup number and status at delivery
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

Pup viability/mortality
In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays. Dead pups were evaluated by the methods, which are described in detail in “Necropsy observations”.

The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7, 8-14 and 15-21 (lactation period) were determined; however, pups, which died accidentally or had to be sacrificed due to maternal death, were not included in these calculations. The number of live pups/litter was calculated on the day after birth, and on lactation days 4, 7, 14, and 21. Furthermore, viability and lactation indices were calculated according to the following formulas:

Viability index (%) = number of live pups on day 4* after birth/number of live pups on the day of birth x 100
* before standardization of litters (i.e. before culling)

Lactation index (%) = number of live pups on day 21 after birth/number of live pups on day 4* after birth x 100
* after standardization of litters (i.e. after culling)


Sex ratio
On the day of birth (PND 0) the sex of the pups was determined by observing the distance between the anus and the base of the genital tubercle; normally, the anogenital distance is considerably greater in male than in female pups. Later, during the course of lactation, this initial sex determination was followed up by surveying the external appearance of the anogenital region and the mammary line. The sex of the pups was finally confirmed at necropsy.

The sex ratio was calculated at PND 0 and PND 21 according to the following formula:

Sex ratio = number of live male or female pups on PND 0 and 21/number of live male and female pups on PND 0 and 21 x 100

Pup necropsy observations

On PND 4, as a result of standardization, selected F1 pups were sacrificed by decapitation under isoflurane anesthesia and blood was sampled for determination of serum thyroid hormone concentrations.
After a similar standardization on PND 4, the surplus F2 pups were sacrificed under isoflurane anesthesia with CO2. After sacrifice, these pups were examined externally, eviscerated and their organs were assessed macroscopically.
On PND 22, the surplus F1 generation pups that were not used for the formation of the cohorts or any investigations were sacrificed under isoflurane anesthesia with CO2 and were examined in the pathology lab. The selected pups for hormone analyses were sacrificed by decapitation under isoflurane anesthesia in the pathology lab and blood was sampled for thyroid hormone analyses .
Pups showing clinical symptoms or gross-morphological findings were examined using appropriate methods. Organs/tissues with gross-morphological findings were preserved in a suitable manner for potential histopathological examination.
Pups that died or were sacrificed in a moribund state were eviscerated and examined for possible defects and/or the cause of death using appropriate methods. These animals were preserved for this purpose, if necessary.
All F1 which were not used for other purposes without any notable findings were discarded after their macroscopic evaluation.

Pup clinical observations

The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams. If pups showed particular findings, these were documented with the dam concerned.

Nipple/areola anlagen
All surviving male pups were examined for the presence of nipple/areola anlagen on PND 13 and were re-examined on PND 20. The number of nipple/areola anlagen were counted.

Pup body weight data
The pups were weighed on the day after birth (PND 1) and on PND 4 (before standardization), 7, 14 and 21.
Pups' body weight change was calculated from these results.
The individual weights were always determined at about the same time of the day (in the morning) and on PND 4 immediately before standardization of the litters.
In the summary tables pup body weights and pup body weight gains are listed for males, females and males + females.

Anogenital distance
Anogenital distance (AGD) is defined as the distance from the center of the anal opening to the base of the genital tubercle. AGD was determined in all live male and female pups on PND
1. These measurements were performed in randomized order, using a measuring ocular. They were conducted by technicians unaware of treatment group in order to minimize bias.

Anogenital index
The anogenital index was calculated according to the following formula:

anogenital index = anogenital distance [mm]/cubic root of pup weight [g]


Sexual maturation

Vaginal opening
All female F1 pups selected to become the F1 rearing animals (cohort 1A and 1B) were evaluated daily for vaginal patency beginning on PND 27. On the day of vaginal opening the body weights of the respective animals were determined.

Preputial separation
All male F1 pups selected to become the F1 rearing animals (cohort 1A and 1B) were evaluated daily for preputial separation beginning on PND 38. On the day of preputial separation the body weights of the respective animals were determined.

Postmortem examinations (parental animals):

Pathological examinations of F0 generation parental animals and F1 generation, rearing animals, cohort 1A

Necropsy
All F0 generation parental animals and all F1 generation, rearing animals, cohort 1A were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention being given to the reproductive organs.)
The following animals died intercurrently (Nos. 58, 322, 342, 377) and were necropsied and assessed by gross pathology as soon as possible after their death.

Organ weights
The following weights were determined in all animals sacrificed on schedule:

1. Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Brain
4. Cauda epididymis
5. Epididymides
6. Heart
7. Kidneys
8. Liver
9. Lymph nodes, axillary (10 animals per sex per group, cohort 1A animals only)
10. Lymph nodes, mesenteric (10 animals per sex per group, cohort 1A animals only)-
11. Ovaries
12. Pituitary gland (fixed)
13. Prostate (ventral and dorsolateral part together, fixed)
14. Testes
15. Seminal vesicles including coagulating glands (fixed)
16. Spleen
17. Thymus (fixed)
18. Thyroid glands (with parathyroid glands) (fixed)
19. Uterus with cervix

All paired organs were weighed together (left and right).

Organ/tissue fixation
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution:

1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymis, left (fixed in modified Davidson´s solution)
11. Esophagus
12. Eyes with optic nerve (fixed in modified Davidson’s solution)
13. Heart
14. Ileum
15. Jejunum (with Peyer’s patches)
16. Kidneys
17. Liver
18. Lungs
19. Lymph nodes, axillary
20. Lymph nodes, mesenteric
21. Mammary gland (male and female)
22. Ovaries (fixed in modified Davidson´s solution)
23. Oviducts
24. Pancreas
25. Pituitary gland
26. Prostate
27. Rectum
28. Sciatic nerve
29. Seminal vesicles
30. Skeletal muscle
31. Spinal cord (cervical, thoracic and lumbar cord)
32. Spleen
33. Stomach (forestomach and glandular stomach)
34. Testis, left (fixed in modified Davidson ´s solution)
35. Thymus
36. Thyroid glands (with parathyroid glands)
37. Trachea
38. Urinary bladder
39. Uterus
40. Vagina
41. Vas deferens

The epididymides, ovaries, testes and eyes with optic nerve of animals that died or were sacrificed intercurrently were fixed in 4% neutral buffered formaldehyde solution.
The left testis and left epididymis of all male F0 parental and Cohort 1A animals sacrificed at scheduled dates were fixed in modified Davidson’s solution, whereas the right testis and epididymis were used for sperm parameters analysis.
For technical reasons, after about 24 hours fixation the ovaries of all F0 and cohort 1A females of all test groups were transferred to 70% ethanol.
The uteri of all cohabited female F0 generation parental animals were examined for the presence and number of implantation sites. The uteri of apparently nonpregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI's method (1)). Then the uteri were rinsed carefully in physiologic salt solution (0.9 % NaCl). When the examinations were completed, the uteri were transferred to the Pathology Laboratory for further processing.

Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the table below:

Organs of F0 and Cohort Test groups
1A animals 00/10 01/11 02/12 03/13

1. All gross lesions A2 A2 A2 A2
2. Adrenal glands A1/A3 A3 A3 A1/A3
3. Bone marrow (femur) A1 A1
4. Brain A1 A1
5. Cecum A1 A1
6. Cervix A1/A3 A3 A3 A1/A3
7. Coagulating glands A1/A3 A3 A3 A1/A3
8. Colon A1 A1
9. Duodenum A1 A1
10. Epididymis, left A1/A3 A3 A3 A1/A3
11. Esophagus A1 A1
12. Eyes with optic nerve A1 A1
13. Heart A1 A1
14. Ileum A1 A1
15. Jejunum A1 A1
16. Kidneys A1 A1
17. Liver A1 A1
18. Lungs A1 A1
19. Lymph nodes, axillary A1 A1
20. Lymph nodes, mesenteric A1 A1
21. Mammary gland (male and female) A1 A1
22. Ovaries A1/A3 A3 A3 A1/A3
23. Oviducts A1/A3 A3 A3 A1/A3
24. Pancreas A1 A1
25. Parathyriod glands A1 A1
26. Pituitary gland A1/A3 A3 A3 A1/A3
27. Prostate A1/A3 A3 A3 A1/A3
28. Peyer’s patches A1 A1
29. Rectum A1 A1
30. Sciatic nerve A1 A1
31. Seminal vesicles A1/A3 A3 A3 A1/A3
32. Skeletal muscle A1 A1
33. Spinal cord (cervical, thoracic, lumbar) A1 A1
34. Spleen A1 A1
35. Stomach (forestomach and glandular stomach) A1 A1
36. Testis, left A1/A3 A3 A3 A1/A3
37. Thymus A1 A1
38. Thyroid glands A1 A1
39. Trachea A1 A1
40. Urinary bladder A1 A1
41. Uterus A1/A3 A3 A3 A1/A3
42. Vagina A1/A3 A3 A3 A1/A3
43. Vas deferens A1/A3 A3 A3 A1/A3

A = Hematoxylin and Eosin (H&E) stain
1 = F0: 20 animals per sex (first 20 surviving pairs with offspring) and all F1A animals/test group 2 = all animals affected/test group
3 = mating pairs suspected of reduced fertility (all F0 animals/test group)

The organs were trimmed according to the “Revised guides for organ sampling and trimming in rats and mice” (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004).
For technical reasons, the left testis, left epididymis, and the eyes with optic nerves of all animals of the F0 generatoin parental control and high dose groups sacrificed at scheduled dates were embedded in paraplast after fixation. For the same reason, the ovaries of all F0 generation parental animals and F1 generation, rearing females, cohort 1A in all test groups will be embedded in paraplast.
A correlation between gross lesions and histopathological findings was attempted. Special attention was given to stages of spermatogenesis in the male gonads.
Special attention was also given to the synchrony of the morphology in ovaries, uterus, cervix, and vagina to the estrous cycle status.
Animals that died or were sacrificed in a moribund state were processed histotechnically and assessed like control animals.
Reproductive organs of all F0 generation parental animals suspected of reduced fertility were subjected to histopathological investigation.

Differential Ovarian Follicle Count (DOFC) in F1 generation, rearing females, cohort 1A
A differential ovarian follicle count (DOFC) was conducted in test groups 10 and 13 (cohort 1A females) according to Plowchalk et.al. (1993). In general, sections were prepared with 2 - 3 µm thickness and the serial sections were taken every 100 µm. For the counting of primordial and growing follicles H&E stained slides were prepared.
Whenever in the ovary the diagnosis: ”no abnormalities detected” was used that implies that all different stages of functional bodies (especially corpora lutea) were present and normal.

Postmortem examinations (offspring):

Pathological examinations of F1 generation, cohort 1B animals

Necropsy
All F1 generation, rearing animals, cohort 1B were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology; special attention was given to the reproductive organs. Animals which died (No. 457) intercurrently were necropsied as soon as possible after their death and assessed by gross pathology.

Organ weights
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Adrenal glands (fixed)
3. Cauda epididymis
4. Epididymides
5. Liver
6. Ovaries
7. Pituitary gland (fixed)
8. Prostate (ventral and dorsolateral part together, fixed)
9. Testes
10. Seminal vesicles including coagulating gland (fixed)
11. Uterus (with cervix)

All paired organs were weighed together (left and right)

Organ/Tissue fixation
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Cervix uteri
4. Coagulating glands
5. Epididymis, left (fixed in modified Davidson ´s solution)
6. Liver
7. Ovaries (fixed in modified Davidson´s solution)
8. Pituitary gland
9. Prostate
10. Seminal vesicles including coagulating glands
11. Testis, left (fixed in modified Davidson ´s solution)
12. Uterus
13. Vagina

The testes and epididymides of the animal that died (No. 457) were fixed in 4% buffered formaldehyde solution.

Histopathology
Histotechnical processing and examination by light microscopy was not performed
For technical reasons, the ovaries of all cohort 1B females of all test groups were embedded in paraplast.


Pathological examinations of surplus F1 generation pups on PND 22 (F1 weanlings not selected for cohorts)

Necropsy
All surplus F1 generation pups that were not used for the following organ weight determinations were sacrificed under isoflurane anesthesia with CO2. The selected pups for organ weight determination were sacrificed by decapitation under isoflurane anesthesia. All animals were necropsied and assessed by gross pathology with special emphasis on the reproductive organs.

Organ weights
The following weights were determined in up to 10 animals per sex per group sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Brain
3. Spleen
4. Thymus (fixed)

The following organs or tissues of up to 10 animals per sex per group were fixed in 4% neutral- buffered formaldehyde solution:
1. All gross lesions
2. Brain
3. Mammary gland (male and female)
4. Spleen
5. Thymus
6. Thyroid glands

Histopathology
Histotechnical processing and examination was not performed

Statistics:

Statistics of the clinical examinations

Statistical analyses were performed according to following tables (GROSSE System):

Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), estrous cycle duration, number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter, duration of sexual maturation (days to vaginal opening, days to preputial separation), anogenital distance, anogenital index:
Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means

Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, lactation index, number of litters with affected pups at necropsy, sexual maturation data (vaginal opening, preputial separation):
Pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions

Presence of areolae/nipples, proportions of affected pups per litter with necropsy observations:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians

Food consumption (parental and rearing animals), body weight and body weight change (parental and rearing animals):
Simultaneous comparison of all dose groups with the control group using the DUNNETT test (two-sided) for the hypothesis of equal means

Reproductive indices:

Male reproduction data
For the males, mating and fertility indices were calculated for F1 litters according to the following formulas:
Male mating index (%) = number of males with confirmed mating*/number of males placed with females x 100
* defined by a female with vaginal sperm or with implants in utero

Male fertility index (%) = number of males proving their fertility*/number of males placed with females x 100
* defined by a female with implants in utero

Female reproduction and delivery data
The pairing partners, the number of mating days until vaginal sperm were detected, and gestational status were recorded for F0 females.
For the females, mating, fertility and gestation indices were calculated for F1 litters according to the following formulas:

Female mating index (%) = number of females mated*/number of females placed with males x 100
* defined as the number of females with vaginal sperm or with implants in utero

Female fertility index (%) = number of females pregnant*/number of females mated** x 100
* defined as the number of females with implants in utero
** defined as the number of females with vaginal sperm or with implants in utero

Gestation index (%) = number of females with live pups on the day of birth/number of females pregnant* x 100
* defined as the number of females with implants in utero

The total number of pups delivered and the number of liveborn and stillborn pups were noted, and the live birth index was calculated for F1 litters according to the following formula:
Live birth index (%) = number of liveborn pups at birth/total number of pups born x 100

The implantations were counted and the postimplantation loss (in %) was calculated according the following formula:

Postimplantation loss (%) = number of implantations – number of pups delivered/number of implantations x 100

Offspring viability indices:

see "Litter observations"

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical observations for males and females
(except gestation and lactation period)
Transient salivation during a short time period after gavage dosing was noted for all high-dose males and females during the entire study and for one mid-dose male during the premating period. It is likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse finding indicating systemic toxicity.
One low- and one mid-dose female (Nos. 140 and 173, respectively) showed temporary ataxia during study weeks 7 - 8 and 6 - 8, respectively. These individual observations were not considered to be associated with the test compound.

Clinical observations for females during gestation of F1 litters
Transient salivation during a short time period after gavage dosing was noted for nearly all high-dose females during gestation period.It is likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse finding indicating systemic toxicity.
One mid-dose female (No. 173) showed ataxia and tremor on a single occasion at GD 21. This individual observation was not considered to be associated with the test compound.

Clinical observations for females and offspring during lactation of F1 litters
Transient salivation during a short time period after gavage dosing was noted for nearly all high-dose females during lactation period. It is likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse finding indicating systemic toxicity.

One low and mid-dose female (Nos. 140 and 173, respectively) showed ataxia during PND 4 - 21 and 0 - 21, respectively. Further, mid-dose female No. 173 showed tremor during PND 0 -4. One low-dose female (No.142) had no more pups alive on PND 3. These observations were not considered to be associated with the test compound.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no test substance-related mortalities in any of the groups.

One male animal (No. 58) of test group 02 was found dead on study week 2 after premating. The death of this animal was not preceded by any specific preterminal clinical signs.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Body weights and body weight change of all test substance-treated male and female F0 animals were comparable to the concurrent control values throughout the entire study.

The statistically significantly increased body weight change in the high-dose males during premating days 63 – 69, as well as in the mid- and high-dose males during study weeks 1 - 2 after premating, respectively, were considered to be spontaneous in nature and not treatment-related.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption of all male and female animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed.
At the end of the administration period, in parental females of test group 1 (100 mg/kg bw/d) mean corpuscular volume (MCV) was significantly lower compared to controls, and in females of test group 2 (300 mg/kg bw/d) absolute eosinophil counts were significantly increased. However, both alterations were not dose dependent and therefore, they were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed.
In females of test group 01 (100 mg/kg bw/d) aspartate aminotransferase (AST) activity was significantly decreased, but the change was not dose dependent. Therefore, it was regarded as incidental and not treatment related.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related, adverse changes among urinalysis parameters were observed.
At the end of the administration period in males of test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) specific gravity of the urine was significantly decreased whereas urine volume was increased, although not statistically significantly. In males of test groups 2 and 3 transitional epithelial cells were found at a significant higher incidence in the urine sediment compared to controls. Higher urine volume and lower specific gravity of the urine without any change of other urine parameters and no histopathological findings in the urinary tract, were regarded as treatment related, but adaptive rather than adverse. The isolated finding of higher incidences of transitional epithelial cells only in males without any histopathologic correlate was also regarded as maybe treatment related but non-adverse.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.
One male animal (No. 58) of test group 02 was found dead before detailed clinical observations in test week 12. The death of this animal was not preceded by any specific preterminal clinical signs.
One low-dose female (No. 140) showed ataxia during detailed clinical observations weeks 14 – 17. One mid-dose female (No. 173) showed ataxia and tremor during DCO weeks 13 - 17 and during DCO week 13, respectively.
All these observations were not considered to be associated with the test compound.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Fertility
The female animal (No. 118), which was not pregnant as well as its male mating partner (No. 18) did not show relevant histopathological findings consistent with impaired fertility.

Decedents
Male animal No. 58 was found dead. No macroscopic or microscopic findings were observed that could explain the death of this animal.

Other effects:

no effects observed

Description (incidence and severity):

Thyroid hormones
In parental males and females (test groups 1, 2 and 3; 100, 300 and 1000 mg/kg bw/d) no treatment-related alterations of T4 and TSH levels were observed.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle data, generated during the last 3 weeks prior to mating to produce the F1 litter, revealed regular cycles in the females of all test groups including the control. The mean estrous cycle duration was similar: 3.9 / 4.1 / 3.9 and 3.9 days in test groups 00 - 03, respectively.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis of parental males no treatment-related effects were observed.

Reproductive performance:

no effects observed

Description (incidence and severity):

Male reproductive data
For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all test groups.
Fertility was proven for most of the F0 parental males within the scheduled mating interval for F1 litter.
One control male (No. 18) did not generate F1 pups. This animal did not show relevant gross or histopathological findings consistent with impaired fertility.
Thus, the male fertility index ranged between 96% and 100%, reflecting the normal range of biological variation inherent in the strain of rats used for this study.

Female reproduction and delivery data
The female mating index calculated after the mating period for F1 litter was 100% in all test groups.
The mean duration until copulation was detected (GD 0) varied between 2.2 and 3.0 days without any relation to dosing.
All female rats delivered pups or had implants in utero with the following exception:
•Test group 00: female No. 118 (mated with male No. 18) did not become pregnant
This animal did not show relevant gross or histopathological findings consistent with impaired fertility.

The fertility index ranged between 96% and 100% without showing any relation to dosing. The mean duration of gestation was similar in all test groups (i.e. between 22.2 and 22.3 days). The gestation index was 100% in in all test groups.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (12.4 / 12.7 / 12.6 and 12.1 implants/dam in test groups 00 - 03, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any statistically significant differences between the groups (7.7 / 9.0 / 8.8 and 8.9 mean% in test groups 00 - 03, respectively), and the mean number of F1 pups delivered per dam remained unaffected (11.4 / 11.6 / 11.4 and 11.1 pups/dam, respectively in test groups 00 - 03).
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 99% / 97% / 99% and 100% in test groups. Moreover, the number of stillborn pups was not significantly different between the test groups.

Thus, Reaction mass of 2-methylbutyl acetate and pentyl acetate did not adversely affect reproduction and delivery of the F0 generation parental females.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

F1 generation pups/litters

There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.

F1 rearing animals, Cohort 1A

Transient salivation during a short time period after gavage dosing was noted for nearly all high-dose males and females during the entire study. It is likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse finding indicating systemic toxicity.
No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.

F1 rearing animals, Cohort 1B

Transient salivation during a short time period after gavage dosing was noted for nearly all high-dose males and females during the entire study. It is likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse finding indicating systemic toxicity.
No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

F1 generation pups/litters

The viability index indicating pup survival during early lactation (PND 0 - 4) varied between 99% / 94% / 100% and 99% in test groups 00 - 03.
The statistically significantly reduced number of surviving pups as well as increased number of cannibalized pups during PND 0 - 4 in test group 01 was caused by female No 142 which had no more live pups on PND 3. This single incident is considered to be spontaneous in nature and not treatment-related.
The lactation index indicating pup survival on PND 4 - 21 was 100% / 100% / 98% and 100%
in test groups 00 - 03.

F1 rearing animals, Cohort 1A

There were no test substance-related mortalities in any of the groups.
One female animal (No. 342) of test group 12 was found dead on study day 8 (accidental death, partly cannibalized) and one female animal (No.377) of test group 13 died due to a gavage error on study day 7.

F1 rearing animals, Cohort 1B

There were no test substance-related mortalities in any of the groups.
One male animal (No. 457) of test group 12 died due to a gavage error on study day 0.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

F1 generation pups/litters

The mean body weights and body weight change of all male and female pups in all test substance-treated groups were comparable to the concurrent control values throughout the entire study.

F1 rearing animals, Cohort 1A

Mean body weights of the low-dose males and females were below the concurrent control values already at the beginning of treatment. They remained below control during the rest of the study. As the difference to the control did not change as treatment progressed this is considered to be an incidental finding.
Mean body weights were comparable to the concurrent control values in the mid- and high- dose males and females during the entire study.
Body weight change was statistically significantly below the concurrent control values for the high-dose males during study days 7 - 14 and for the low-dose males during study days 7 - 21. In the low- and high-dose females body weight change was statistically significantly increased during study days 35 – 42 and in turn decreased during study days 42 - 49. As all these changes were not consistent and followed no dose-response they are considered to be chance findings rather than associated with the treatment.
The body weight change of the mid-dose male and female rats were comparable to the concurrent control values throughout the entire study.

F1 rearing animals, Cohort 1B

Body weights of all test substance-treated male as well as of mid- and high-dose females animals were comparable to the concurrent control values throughout the entire study. The body weight change of the mid- and high-dose male and female rats were comparable to the concurrent control values throughout the entire study.
Mean body weights of the low-dose females were statistically significantly below the concurrent control values on study day 7, during days 21 - 35 and on day 49. Body weight change was statistically significantly below the concurrent control values for the low-dose males during study days 7 - 14 and for the low-dose females during study days 14 - 21 and 42 - 49. As all these changes were not consistent and followed no dose-response they are considered to be chance findings rather than associated with the treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

F1 rearing animals, Cohort 1A

Food consumption was comparable to the concurrent control values in the mid-dose males and in the mid- and high-dose females during the entire study.
Food consumption of the high-dose males was statistically significantly below the concurrent control values during study days 14 - 21, 28 - 35, and 49 - 56 (about 6 - 8%). Food consumption of the low-dose males was statistically significantly below the concurrent control values during study days 7 - 49 (about 7 - 10%). Food consumption was statistically significantly below the concurrent control values for the low-dose females during study days 7 - 14 (about 5%). As all these changes were minor and followed no dose-response they are considered to be chance findings rather than associated with the treatment.

F1 rearing animals, Cohort 1B

Food consumption of all male and female animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed.
At the end of the administration period in F1A males of test groups 12 (300 mg/kg bw/d) prothrombin time (i.e., Hepatoquick’s test, HQT) was significantly shortened, and absolute large unstained cell (LUC) counts were significantly increased. In males of test groups 12 and 13 (300 and 1000 mg/kg bw/d) relative eosinophil counts were significantly decreased. In F1A females of test group 11 (100 mg/kg bw/d) total white blood cell (WBC), absolute eosinophil, neutrophil and lymphocyte counts were significantly higher compared to controls. This was also true for WBC and absolute neutrophil counts in females of test group 12 (300 mg/kg bw/d and absolute neutrophil counts in females of test group 13 (1000 mg/kg bw/d). However, all mentioned alterations were not dose dependent and therefore, they were regarded as incidental and not treatment related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed.
At the end of the administration period in F1A males of test groups 12 and 13 (300 and 1000 mg/kg bw/d) inorganic phosphate levels were significantly increased, but this alteration was not dose dependent. Therefore, it was regarded as incidental and not treatment related.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among urinalysis parameters were observed.
In F1 females of test group 11 (100 mg/kg bw/d) urine pH values were significantly increased. However, this alteration was not dose-dependent and therefore, it was regarded as incidental and not treatment related.

Sexual maturation:

no effects observed

Description (incidence and severity):

F1 generation pups/litters

Vaginal opening
Each female F1 pup, which was selected to become a rearing female, was evaluated for commencement of sexual maturity. The first day when vaginal opening was observed was PND 27, the last was PND 38. The mean number of days to reach the criterion in the control and 100, 300 and 1000 mg/kg bw/d test groups was 31.4; 32.0; 31.4 and 31.2 days, respectively. The mean body weight on the day, when vaginal opening was recorded, amounted to 98.3 g,
98.5 g, 99.9 g and 98.3 g in test groups 00 - 03. Neither a statistically significant nor a toxicologically relevant effect was noted in any of the treatment groups.

Preputial separation
Each male F1 pup, which was selected to become a rearing male, was evaluated for commencement of sexual maturity. The first day when preputial separation was observed was PND 38, the last was PND 51. The mean number of days to reach the criterion in the control and 100, 300 and 1000 mg/kg bw/d test groups was 41.4, 42.2, 41.9 and 41.5 days, respectively. The mean body weight on the day, when preputial separation was recorded, amounted to 176.4 g, 173.0 g, 179.0 g and 174.6 g in test groups 00 - 03. Neither a statistically significant nor a toxicologically relevant effect was noted in any of the treatment groups.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

F1 generation pups/litters
The anogenital distance and anogenital index of all test substance treated male and female pups was comparable to the concurrent control values.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

F1 generation pups/litters
The percentage of male pups having nipples/areolae was not influenced by the test substance when examined on PND 13.
During the re-examination at weaning no nipples/areolae were detected in any male pup of all test groups.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Surplus F1 generation pups on PND 22 (F1 weanlings not selected for cohorts)

Absolute organ weights
All mean absolute weight parameters did not show significant differences when compared to the control group 00.

Relative organ weights
All mean relative weight parameters did not show significant differences when compared to the control group 00.

F1 rearing animals, Cohort 1A

Absolute organ weights
When compared with control group 10 (=100%), the following mean absolute weights were significantly changed:

F1A Male animals Female animals
Test group 11 12 13 11 12 13
(mg/kg bw/d) (100) (300) (1000) (100) (300) (1000)
Final body weight 92%** 97% 94%*
Kidneys 93%* 100% 94%
Liver 89%** 96% 88%*
Pituitary gland 92% 95% 93%**
Spleen 99% 101% 91%*
Thymus 94% 104% 86%**
Thyroid glands 103% 87%* 102%
* : p <= 0.05, **: p <= 0.01

All other mean absolute weight parameters did not show significant differences when compared to the control group 10.

Relative organ weights
When compared with control group 10 (=100%), the following mean relative weights were significantly changed:

F1A Male animals Female animals
Test group 11 12 13 11 12 13
(mg/kg bw/d) (100) (300) (1000) (100) (300) (1000)
Brain 106%** 102% 104%*
Epididymides 108%** 105%* 106%*
Thymus 103% 106% 91%*
Thyroid glands 107% 85%* 102%
* : p <= 0.05, **: p <= 0.01

All other mean relative weight parameters did not show significant differences when compared to the control group 10.

The significantly reduced final body weight in test group 11 and 13 (100 and 1000 mg/kg bw/d) in males was regarded to be incidental. The weight of test group 11 was slightly below historical control values but the body weight of test group 13 males was within historical control values. Therefore, these changes in final body weight were regarded to be incidental and unrelated to treatment. All other absolute and relative weight changes in male animals were thought to be secondary to the body weight changes and consequently unrelated to treatment.


F1 rearing animals, Cohort 1B

Absolute and relative organ weights
When compared with control group 10 (=100%), the following mean absolute weights were significantly changed:

F1B Male animals Female animals
Test group 11 12 13 11 12 13
(mg/kg bw/d) (100) (300) (1000) (100) (300) (1000)
Adrenal glands 93% 97% 91%*
Epididymides 96%* 100% 97%
* : p <= 0.05, **: p <= 0.01

All other mean absolute weight parameters did not show significant differences when compared to the control group 10.

Relative organ weights
When compared with control group 10 (=100%), the following mean relative weights were significantly changed:

F1B Female animals
Test group 11 12 13
(mg/kg bw/d) (100) (300) (1000)
Ovaries 100% 106%** 96%
* : p <= 0.05, **: p <= 0.01

All other mean relative weight parameters did not show significant differences when compared to the control group 10.

The slight significant decrease of absolute weight of epididymides of test group 11 (100 mg/kg bw/d) animals was considered to be incidental and a consequence of the not-statistically reduced final body weight (-3%).

The decrease of absolute adrenal gland weight in females of test group 13 (1000 mg/kg bw/d) was within historical control data and was therefore not regarded to be treatment related. The same comes true for the increased relative ovaries weight of test group 12 (300 mg/kg bw/d) animals and was also considered as incidental.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Surplus F1 generation pups on PND 22 (F1 weanlings not selected for cohorts)

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

F1 generation pups/litters

A few F1 pups showed spontaneous findings at gross necropsy, such as post mortem autolysis, discolored thymus, expanded lung, empty stomach, stomach erosions and discolored skin.
These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were not considered to be associated with the test substance.

F1 rearing animals, Cohort 1A

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

F1 rearing animals, Cohort 1B

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Decedents
The animal No. 457, showed dilation of the lungs.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F1 rearing animals, Cohort 1A

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Decedents
Female animal No. 322 was found dead. It showed grey-yellow deposition on heart and pericard. By light microscopy, inflammation of the pericard, the trachea, the capsules of thymus and thyroid glands was observed. These findings were most likely caused by a gavage error. The female animals (No. 342 and 377) that died did not show relevant macroscopic or microscopic findings that could have caused their death.

Differential ovarian follicle count
The results of the differential ovarian follicle count (DOFC) – comprising the numbers of primordial and growing follicles, as well as the combined incidence of primordial plus growing follicles – did not reveal significant differences between the control group 10 and animals of test group 13 (1000 mg/kg bw/d):

Number Absolute values
of animals Group Primordial Growing Primordial + growing
20 10 4672 574 5246
19 13 4878 615 5493
* : p < 0.05 ** = p < 0.01

Number Mean values
of animals Group Primordial Growing Primordial + growing
20 10 233.60 28.70 262.30
19 13 256.73 32.37 289.10
* : p < 0.05 ** = p < 0.01

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

F1 generation pups/litters

Pup number and status at delivery
The mean number of delivered F1 pups per dam and the rates of liveborn and stillborn F1 pups were evenly distributed about the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.

Sex ratio
The sex distribution and sex ratios of live F1 pups on the day of birth and on PND 21 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.

Thyroid hormones
In the F1 PND4 and PND22 pups (test groups 1, 2 and 3; 100, 300 and 1000 mg/kg bw/d) no treatment-related alterations of T4 and TSH levels were observed

F1 rearing animals, Cohort 1A

Detailed clinical observations
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.
One female animal (No. 342) of test group 12 had an accidental death in DCO week 1 and one female animal (No. 377) of test group 13 died after gavage error in DCO week 1.

Estrous cycle data
Estrous cycle data, generated during 2 weeks, revealed regular cycles in the females of all test groups 10 - 13. The mean estrous cycle duration was similar: 4.1 / 4.0 / 4.0 and 4.0 days in test groups 10 - 13, respectively.

Thyroid hormones
In F1A males and females (test groups 11, 12 and 13; 100, 300 and 1000 mg/kg bw/d) no treatment-related alterations of T4 and TSH levels were observed.

Spermanalysis
Concerning motility of the sperms and the incidence of abnormal sperms in the cauda epididymidis as well as sperm head counts in the testis and in the cauda epididymidis of F1A males no treatment-related effects were observed.


F1 rearing animals, Cohort 1B

Detailed clinical observations
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.
One male animal (No. 457) of test group 12 died after gavage error in DCO week 0.

Estrous cycle data
Estrous cycle data, generated during 2 weeks, revealed regular cycles in the females of all test groups 10 - 13. The mean estrous cycle duration was similar: 4.0 / 4.2 / 4.0 and 4.0 days in test groups 10 - 13, respectively.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Reproductive effects observed:

no

Conclusions:

Under the conditions of the present extended one-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicity is 1000 mg/kg bw/d, the highest tested dose.
The NOAEL for fertility and reproductive performance for the parental rats is 1000 mg/kg bw/d, the highest tested dose.
The NOAEL for developmental toxicity in the F1 progeny is 1000mg/kg bw/d, the highest tested dose.

Executive summary:

Reaction mass of 2 -methylbutyl acetate and pentyl acetate was administered to groups of 25 male and 25 female healthy young Wistar rats as test groups 00 - 03 as an aqueous preparation by stomach tube at different dosages(0, 100, 300 and 1000 mg/kg body weight/day [mg/kgbw/d]).F0 animals were treated at least for 10 weeks prior to mating to produce a litter (F1 generation). Mating pairs were from the same dose group. Pups of the F1 litter were selected (F1 rearing animals) and assigned to 2 different cohorts (1A, 1B) which were subjected to specific postweaning examinations. The study was terminated with the terminal sacrifice of the F1 rearing animals of cohort 1A. Control animals were dosed daily with the vehicle (0.5% CMC suspension in deionized water with Cremophor EL [10 mg/100mL]).

 

In general, analyses confirmed the prepared concentrations, the homogeneous distribution and the stability of the test substance in the vehicle.

 

There were no test substance-related mortalities or adverse clinical observations, indicating systemic toxicity, noted in any of the groups. In particular,regularly conducted detailed clinical observations revealed no test substance-related adverse effects.Transient salivation during a short time period after gavage dosing was noted for nearly all high-dose animals during all sections of the study. It is likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse finding indicating systemic toxicity.

 

Food consumption was essentially comparable to the concurrent control values in all F0, F1A and F1B dose groups throughout the study. Some minor and patchy changes which followed no dose-response were considered to be chance findings rather than associated with the treatment.

 

Body weights and body weight change of all test substance-treated male and female F0, F1A and F1 Brats were essentially comparable to the concurrent control values throughout the entire study. Some minor and patchy changes which followed no dose-response were considered to be chance findings rather than associated with thetreatment.

 

Concerning clinical pathology, in F0 as well as F1 rats no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d.

 

Regarding pathology, no test substance-related adverse findings were noted in any of the dose groups.

 

F0 GENERATION PARENTAL ANIMALS

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

 

The reproductive organs of the mating pair suspected of reduced fertility did not show histopathological findings that could explain the reduced fertility.

 

F1 GENERATION, REARING ANIMALS, COHORT 1A

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation totreatment.

 

The results of the differential ovarian follicle count (DOFC) – comprising the numbers of primordial and growing follicles, as well as the combined incidence of primordial plus growing follicles–showed no significant differences between the placebo group 10 and animals of test group13.

 

F1 GENERATION, REARING ANIMALS, COHORT 1B

One animal of this cohort died. Beside of dilated lungs, no gross finding was noted that could explain the death in this animal.

 

SURPLUS F1 GENERATION PUPS ON PND 22 (F1 WEANLINGS NOT SELECTED FOR COHORTS)

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

 

There were no indications from clinical examinations as well as gross and histopathology, that Reaction mass of2 -methylbutyl acetate and pentyl acetate adversely affected the fertility or reproductiveperformance of the F0 parental animals up to and including the administered high-dose of 1000 mg/kg bw/d. Estrous cycle data, sperm quality of males, mating behavior, conception, gestation, parturition, lactation and weaning as well as sexual organ weights and gross and histopathological findings of these organs (specifically the differential ovarian follicle count)were comparable between the rats of all groups including control and ranged within the historical control data of the test facility.

 

For all liveborn male and female pups of the F0 parents, no test substance-induced signs of developmental toxicity were noted at dose levels as high as 1000 mg/kg bw/d. Postnatal survival, pup body weight gain as well as post-weaning development of the offspring of this test group until puberty remained unaffected by the test substance. Furthermore, clinical and/or gross necropsy examinations of the F1 pups revealed no adverse findings.

 

Measurement of thyroid hormones revealed no effect caused by the test substance, neither in the F0 parental animals nor in the liveborn F1 offspring.

 

Neither the anogenital distance/index nor the check for the presence of nipples/areolas, both very sensitive marker of potential endocrine-mediated imbalances, revealed any test substance-related effects.

 

Vaginal opening and preputial separation are commonly used developmental markers for onset of pubertyin laboratory rats. No delays beyond a normal range of biological variation in rat (multi)generation studies which might be attributable to the treatment were noted in any of the test substance-treated groups.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Feb 2019 - 16 Oct 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Justification for study design:

Basis for dose level selection:
In a preliminary screening study with wistar rats no adverse effects were observed up to the limit dose (1000 mg/kg bw/d).

Specific details on test material used for the study:

The various analyses:
• Demonstrated the stability of the test substance preparations over a period of 7 days at room temperature (including stability over 3 days in the refrigerator).
• Confirmed the homogeneous distribution of the test substance in 0.5 % CMC suspension in deionized water (with 10 mg/100 mL Cremophor EL)
• Confirmed the overall accuracy of the prepared test substance concentrations

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female Wistar rats, strain Crl:WI(Han), supplied by Charles River Laboratories, Research Models and Services, Germany GmbH which were free from any clinical signs of disease, were used for the investigations. The females were nulliparous and non-pregnant at the beginning of the study. The receipt of males (about 77 - 90 days old) and females (about 70 - 76 days old) at different age warrants that no sibling males and females will be paired during the study. These animals were used as F0 generation parental animals. All other animals used in this study (F1 generation pups) were derived from the supplier-provided animals.

HOUSING AND DIET
During the pretreatment period of the study, the rats were housed together (up to 5 animals per sex and cage) in Polysulfonate cages Typ 2000P (H-Temp) supplied by TECHNIPLAST, Hohenpeißenberg, Germany.
During the study period, the rats were housed individually in Polycarbonate cages type III supplied by TECHNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany, with the following exceptions:
• During overnight matings, male and female mating partners were housed together in Polycarbonate cages type III.
• Pregnant animals and their litters were housed together until PND 13 in Polycarbonate cages type III.
Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
For enrichment wooden gnawing blocks (Typ Lignocel® block large, new name: SAFE® block large J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany) were added. In addition in Polysulfonate cages large play tunnels (Art. 14153; supplied by PLEXX B.V., Elst, Netherlands) were added. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured. The animals were housed in fully air-conditioned rooms in which central air conditioning guaranteed a range of temperature of 20-24°C and a range of relative humidity of 45-65%. The air change rate was 15 times per hour. There were no or only minimal deviations from these limits. The day/night cycle was 12 hours light from 6.00 h to 18.00 h and 12 hours darkness from 18.00 h to 6.00 h. The animal room was completely disinfected using a disinfector ("AUTEX" fully automatic, formalin-ammonia-based terminal disinfection) before use. Walls and floor were cleaned once a week with water containing an appropriate disinfectant. The food used was ground Kliba maintenance diet mouse/rat “GLP” meal, supplied by Garanovit AG, Kaiseraugst, Switzerland, which was available to the animals ad libitum throughout the study (from the day of supply to the day before necropsy). Drinking water was supplied from water bottles (ad libitum). Dust-free wooden bedding was used in this study.

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

Test substance preparations in 0.5% CMC suspension in deionized water (with 10 mg/ 100 mL Cremophor EL)

Details on exposure:

The test substance suspensions in 0.5% CMC suspension in deionized water with 10 mg/100 mL Cremophor EL were prepared in intervals, which took into account the analytical results of the stability verification. For the preparation of the administration suspensions the specific amount of test substance was weighed in a calibrated Erlenmeyer flask, topped up with cooled 0.5% CMC suspension in deionized water with 10 mg/100 mL Cremophor EL and intensely mixed with the magnetic stirrer. Afterwards the test substance preparations were kept cool in the refrigerator. For the daily administration, the test substance preparations were filled in smaller portions which were kept homogeneous with a magnetic stirrer at room temperature. During the administration the Erlenmeyer flask was closed with a plastic plug with a small hole and the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
According to preliminary test substance preparation analyses, the test substance is not homogenously soluble in water, therefore 0.5% CMC suspension in deionized water with Cremophor EL (10 mg/100mL) was chosen as solubilising agent

Details on mating procedure:

The male and female rats were about 10 - 12 weeks old, when they arrived from the breeder. During an acclimatization period of about 3 weeks, estrous cycle determination prior to treatment was performed in a pool of up to 50 non-randomized female animals. Animals with no regular estrous cycle and/or animals with the lowest and highest body weights were eliminated from the study and used for other purposes. The 40 male and 40 female animals included in the study were about 13 - 15 weeks old at the beginning of treatment and their weight variation did not exceed 20 percent of the mean weight of each sex. In general, each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same dose group. The animals were paired by placing the female in the cage of the male mating partner from about 16.00 h until 6.30 - 9.00 h of the following morning. Deviations from the specified times were possible on weekends and public holidays and were reported in the raw data. A vaginal smear was prepared after each mating and examined for the presence of sperm. If sperm was detected, pairing of the animals was discontinued. The day on which sperm were detected was denoted " gestation day (GD) 0" and the following day "gestation day (GD) 1"

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany. Analytical verifications of the stability of the test substance in 0.5% CMC suspension in deionized water with 10 mg/100 mL Cremophor EL for a period of 7 days at room temperature and refrigerator (+4°C) had been initiated prior to the start of the study. At the beginning (during premating), twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. The 3 samples were withdrawn from the top, middle and bottom of the preparation vessel. These samples were used as a concentration control at the same time. At the above mentioned time points additionally one sample from the mid concentration was taken for concentration control analysis. All test samples, plus a duplicate set of reserve samples, were withdrawn by staff of the Laboratory Reproduction Toxicology. The samples collected at the beginning of the administration period and during lactation were analyzed in the Analytical Laboratory. The samples of the gestation were not analyzed because no relevant imprecision occurs during the analysis of the samples from the beginning and lactation of the study. The samples within this study were labeled with serial numbers. The same number was not used for several samplings. Reserve samples were labeled with 1R, 2R, etc. The reserve samples were stored at the Laboratory Reproduction Toxicology frozen (at -20 °C) Analysis of these samples were performed in case of equivocal analytical results with the original samples or after loss of/damage to original samples after agreement by the Study Director.

RESULTS
The stability of test substance in 0.5% CMC suspension in deionized water (with 10 mg/100 mL Cremophor EL) was demonstrated for a period of 7 days at room temperature (including stability over 3 days in the refrigerator).
The homogeneous distribution of the test substance in 0.5% CMC suspension in deionized water (with 10 mg/100 mL Cremophor EL) was demonstrated.
Measured values for Reaction mass of 2-methylbutyl acetate and pentyl acetate were in the expected range of the target concentrations (90 – 110 %) demonstrating the correctness of the preparations. For the low dose (100 mg/kg bw/d), mean values of sample No. 30 - 32 and their overall mean value of around 89 % at one time point was marginally below the range of 90 - 110 %. However, the test substance preparation was homogeneously distributed for this dose level and all other values of the low dose were clearly within the tolerance range. Therefore, the overall measured values of the low dose were assessed to be in an acceptable range of the nominal concentrations in this study.

Duration of treatment / exposure:

After the acclimatization period, the test substance was administered to the parental animals orally by gavage, once daily at approximately the same time in the morning. Females in labor were not treated. The treatment lasted up to one day prior to sacrifice. The animals of the control group were treated with the vehicle (0.5% CMC suspension in deionized water with 10 mg/100 mL Cremophor EL), in the same way. The volume administered each day was 10 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.

The duration of treatment covered a 30 days in-life period in males (including premating, mating [mating pairs were from the same test group] and postmating period) and a 2-weeks premating and mating period, the entire gestation and approximately 3 weeks of lactation period in females. Parental females were allowed to give birth and bring up the offspring until sacrifice on PND 4 or 13.

The male and female animals were sacrificed 30 and 56 days, respectively, after the beginning of the administration, and examined.

Frequency of treatment:

Once daily

Details on study schedule:

Standardization of litters (F1 generation pups)
On PND 4, the individual litters were standardized in such a way that, where possible, each litter contained 4 male and 4 female pups (always the first 4 pups/sex and litter were taken for further rearing). If individual litters did not have 4 pups/sex, the litters were processed in such a way that the most evenly distributed 8 pups per litter were present for further rearing (e.g., 5 male and 3 female pups). Surplus pups were sacrificed. Standardization of litters was not performed in litters with ≤ 8 pups.

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

Mortality
A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays). If animals were in a moribund state, they were sacrificed and necropsied.

Clinical observations
A cageside examination was conducted at least daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity before the administration as well as within 2 hours and between 2 and 5 hours after the administration. Abnormalities and changes were documented daily for each animal. Individual data of daily observations can be found in the raw data. The parturition and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. Only particular findings (e.g. inability to deliver) were documented on an individual dam basis. On weekdays (except Saturday, Sunday and public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.
The day of parturition was considered the 24-hour period from about 15.00 h of one day until about 15.00 h of the following day.

Water consumption
Generally, water consumption was determined once a week (each time for a period of 3 days) for the male and female parental animals, with the following exceptions:
• Water consumption was not determined after the 2nd premating week (male parental animals)
• Water consumption of the females with evidence of sperm was determined on gestation days (GD) 0-1, 6-7, 13-14 and 19-20.
• Water consumption of the females, which gave birth to a litter was determined for PND 1-2, 3-4, 6-7, 9-10 and 12-13.
Water consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.

Food consumption
Generally, food consumption was determined once a week for male and female parental animals, with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals).
• Food consumption of the females with evidence of sperm was determined on GD 0 - 7, 7 - 14 and 14 - 20.
• Food consumption of the females which gave birth to a litter was determined on PND 1 - 4, 4 - 7, 7 - 10 and 10 - 13.
Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

Body weight data
In general, the body weight of the male and female parental animals was determined once a week at the same time of the day (in the morning) until sacrifice. The body weight change of the animals was calculated from these results.
• During the mating period the parental females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0) and on PND 1, 4, 7, 10 and 13.
Females without positive evidence of sperm, without litter and females after weaning (PND 13) were weighed weekly. These body weight data were solely used for the calculations of the dose volume; therefore, these values are not reported in the summary.

Detailed clinical observations
Detailed clinical observations (DCO) were performed in all animals once prior to the first administration (day 0) and at weekly intervals during the administration period. The examinations started in the morning. The findings were ranked according to the degree of
severity, if applicable. For observation, the animals were removed from their cages by the investigator and placed in a standard arena (50 × 37.5 × 25 cm). The following parameters listed were assessed:
1. Abnormal behavior in “handling”
2. Fur
3. Skin
4. Posture
5. Salivation
6. Respiration
7. Activity/arousal level
8. Tremors
9. Convulsions
10. Abnormal movements
11. Gait abnormalities
12. Lacrimation
13. Palpebral closure
14. Exophthalmos (Protruding eyeball)
15. Assessment of the feces excreted during the examination (appearance/consistency)
16. Assessment of the urine excreted during the examination
17. Pupil size

Functional observation battery
A functional observational battery (FOB) was performed in first surviving 5 male and selected surviving 5 female animals with litter per group at the end of the administration period starting at about 10.00 h. The FOB started in a randomized sequence with passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests. The findings were ranked according to the degree of severity, if applicable. The observations were performed at random.

Home cage observations
The animals were observed in their closed home cages (for a short period: about 10-30 seconds); any disturbing activities (touching the cage or rack, noise) were avoided during these examinations in order not to influence the behavior of the animals. Attention was paid to:
1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Gait
6. Other findings

Open field observations
The animals were transferred to a standard arena (50 × 50 × 25 cm) and observed. The following parameters were examined:
1. Behavior on removal from cage
2. Fur
3. Skin
4. Salivation
5. Nasal discharge
6. Lacrimation
7. Eyes/pupil size
8. Posture
9. Palpebral closure
10. Respiration
11. Tremors
12. Convulsions
13. Abnormal movements/stereotypy
14. Gait
15. Activity/arousal level
16. Feces (appearance/ consistency) within 2 minutes
17. Urine (amount/color) within 2 minutes
18. Rearing within 2 minutes
19. Other findings

Sensory motor tests/Reflexes
The animals were removed from the open field and subjected to following sensory motor or reflex tests:
1. Reaction to an object being moved towards the face (Approach response)
2. Touch sensitivity (Touch response)
3. Vision (Visual placing response)
4. Pupillary reflex
5. Pinna reflex
6. Audition (Startle response)
7. Coordination of movements (Righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (Tail pinch)
11. Other findings
12. Grip strength of forelimbs
13. Grip strength of hindlimbs
14. Landing foot-splay test

Motor activity measurement
The measurement of motor activity (MA) was measured at the end of the administration period in first surviving 5 male and selected surviving 5 female animals with litter per group. Motor activity (MA) was measured from 14:00 h onwards on the same day as the FOB was performed. The examinations were performed using the TSE Labmaster System supplied by TSE Systems GmbH, Bad Homburg, Germany. For this purpose, the animals were placed in new clean polycarbonate cages with a small amount of bedding for the duration of the measurement. Eighteen beams were allocated per cage. The number of beam interrupts were counted over 12 intervals for 5 minutes per interval. The sequence in which the animals were placed in the cages was selected at random. On account of the time needed to place the animals in the cages, the starting time was "staggered" for each animal. The measurement period began when the 1st beam was interrupted and was finished exactly 1 hour later. No food or water was offered to the animals during these measurements and the measurement room was darkened after the transfer of the last animal.

Oestrous cyclicity (parental animals):

For all females in a pool of up to 50 animals, estrous cycle normality was evaluated before the randomization. For a minimum of 2 weeks prior to mating estrous cycle length was evaluated by daily analysis of vaginal smear for all F0 female parental rats. Determination was continued throughout the pairing period until the female exhibited evidence of copulation. At necropsy, an additional vaginal smear was examined to determine the stage of estrous cycle for each F0 female with scheduled sacrifice.

Litter observations:

Pup number and status at delivery
All pups delivered from the F0 parents (F1 litter) were examined as soon as possible on the day of birth to determine the total number of pups, the sex and the number of liveborn and stillborn pups in each litter. At the same time, the pups were also being examined for macroscopically evident changes. Pups, which died before this initial examination, were defined as stillborn pups.

Pup clinical observations
The live pups were examined daily for clinical symptoms (including gross-morphological findings) during the clinical inspection of the dams and documented for each pup.

Pup body weight data
The pups were weighed on the day after birth (PND 1) and on PND 4 (before standardization), 7 and 13. Pups' body weight change was calculated from these results. The individual weights were always determined at about the same time of the day (in the morning) and on PND 4 immediately before standardization of the litters. In the summary tables pup body weights and pup body weight gains are listed for males, females and males + females. “Runts” were defined on the basis of the body weights on PND 1. "Runts" are pups that weigh less than 75% of the mean weight of the respective control pups.

Anogenital distance
Anogenital distance (AGD) is defined as the distance from the center of the anal opening to the base of the genital tubercle. AGD was determined in all live male and female pups on PND 1. These measurements were performed in randomized order, using a measuring ocular. They were conducted by technicians unaware of treatment group in order to minimize bias.

Nipple/areola anlagen
All surviving male pups were examined for the presence of nipple/areola anlagen on PND 13. The number of nipple/areola anlagen was counted.

Postmortem examinations (parental animals):

Necropsy
All parental animals were sacrificed by decapitation under isoflurane anesthesia. The exsanguinated animals were necropsied and assessed by gross pathology, special attention being given to the reproductive organs.

Organ weights
The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (final body weight)
2. Epididymides
3. Ovaries
4. Prostate (ventral and dorsolateral part together, fixed)
5. Seminal vesicles with coagulating glands (fixed)
6. Testes
7. Thyroid glands (with parathyroid glands) (fixed)
8. Uterus with cervix

The following weights were determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for clinical pathological examinations):
1. Adrenal glands (fixed)
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Spleen
7. Thymus (fixed)

All paired organs were weighed together (left and right).

Organ/tissue fixation
The following organs or tissues of all parental animals were fixed in in 4% neutral buffered formaldehyde solution or in modified Davidson’s solution:
1. All gross lesions
2. Adrenal glands
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Esophagus
12. Epididymides (modified Davidson’s solution)
13. Extraorbital lacrimal glands
14. Eyes with optic nerve (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (axillary and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Testes (modified Davidson’s solution)
43. Thymus
44. Thyroid glands
45. Trachea
46. Urinary bladder
47. Uterus
48. Vagina

The uteri of all cohabited female F0 parental animals were examined for the presence and number of implantation sites. The uteri of apparently nonpregnant animals or empty uterus horns were placed in 1% ammonium sulfide solutions for about 5 minutes in order to be able to identify early resorptions or implantations (SALEWSKI E (1964)). Then the uteri were rinsed carefully in physiologic salt solution (0.9 % NaCl). When the examinations were completed, the uteri were transferred to the Pathology Laboratory for further processing.

Postmortem examinations (offspring):

Pup necropsy observations
On PND 4, as a result of standardization, the surplus pups were sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone concentrations. After sacrifice, the pups were examined externally and eviscerated, and the organs were assessed macroscopically. On PND 13, one selected male and one female pup per litter was sacrificed under isoflurane anesthesia by decapitation. Blood was sampled for determination of thyroid hormone
concentrations. Thyroid glands/parathyroid glands were fixed in neutral buffered 4% formaldehyde solution and were transferred to the Pathology Laboratory for possible further processing. The remaining pups were sacrificed under isoflurane with CO2. After sacrifice, all pups were examined externally and eviscerated, and their organs were assessed macroscopically. All stillborn pups and all pups that died before weaning were examined externally, eviscerated and their organs were assessed macroscopically. All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.

Statistics:

Water consumption (parental animals), food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), gestation days, anogenital distance, anogenital index:
Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (twosided) for the hypothesis of equal means

Male and female mating indices, male and female fertility indices, females mated, females delivering, gestation index (females with liveborn pups), females with stillborn pups, females with all stillborn pups:
Pair-wise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions

Mating days until day 0 pc, %postimplantation loss, pups stillborn, %perinatal loss, nipple development:
Pair-wise comparison of the dose group with the control group using the WILCOXON test (one-sided+) with BONFERRONI-HOLM adjustment for the hypothesis of equal medians

Implantation sites, pups delivered, pups liveborn, live pups day x, viability Index, survival index:
Pair-wise comparison of the dose group with the control group using the WILCOXON test (one-sided-) with BONFERRONI-HOLM adjustment for the hypothesis of equal medians

% live male day x, %live female day x:
Comparison of the dose group with the control group was performed using the WILCOXON test (twosided) for the hypothesis of equal medians

Number of cycles and Cycle Length, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity:
Non-parametric oneway analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (twosided) for the equal medians

Reproductive indices:

Male reproduction data

Male mating index (%) = number of males with confirmed mating / number of males placed with females x 100
Male fertility index (%) = number of males proving their fertility / number of males placed with females x 100

Female reproduction and delivery data

Female mating index (%) = number of females mated / number of females placed with males x 100
Female fertility index (%) = number of females pregnant / number of females mated x 100
Gestation index (%) = number of females with live pups on the day of birth / number of females pregnant x 100
Live birth index (%) = number of liveborn pups at birth / total number of pups born x 100
Postimplantation loss (%) =number of implantations – number of pups delivered / number of implantations x 100


Sex ratio = number of live male or female pups on day 0 and 13 / number of live male and female pups on day 0 and 13 x 100
Anogenital index = anogenital distance [mm] / cubic root of pup weight [g]

Offspring viability indices:

In general, a check was made for any dead or moribund pups twice daily on workdays (once in the morning and once in the afternoon) or as a rule, only in the morning on Saturdays, Sundays or public holidays.
The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4, 5-7 and 8-13 were determined. Pups which died accidentally or had to be sacrificed due to maternal death were not included in these calculations. The number of live pups/litter was calculated on the day after birth (PND 0), and on lactation days 4, 7 and 13. Furthermore, viability and survival indices were calculated according to the following formulas:

Viability index (%) = number of live pups on day 4* after birth / number of live pups on the day of birth
* before standardization of litters (i.e. before culling)

Survival index (%) = number of live pups on day 13 after birth / number of live pups on day 4* after birth x 100
* after standardization of litters (i.e. after culling)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All males and females of the high-dose (1000 mg/kg bw/d) and two out of ten males of the middose (300 mg/kg bw/d) showed salivation immediately after dosing (up to 2 hours post dosing) during the treatment period.
No other clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female F0 parental animals in any of the test groups (1 - 3; 100, 300 and 1000 mg/kg bw/d) during the study.
One control female showed vaginal discharge (color: red) on GD 13. This observation was not considered to be associated with the test compound.
No clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any of the male and female animals in any of the groups.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related or spontaneous mortalities in any of the groups.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weights of all male and all female parental animals in all test substance-treated groups were comparable to the concurrent control values during the entire study.
Mean body weight change of the high-dose parental males was statistically significantly below the concurrent control values during in-life days 7 - 13, 21 - 28 and 0 - 28. Since the body weight change was only marginally decreased, it was assessed as not treatment-related. The slight, statistically significant decrease of the mid-dose parental males during in-life days 0 - 7 was assessed as not treatment-related since it was not related to dose.
Mean body weight change of all test substance-treated females and of the mid- and low-dose males was comparable to the concurrent control values during the entire study

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption of all male and all female animals of all test substance-treated groups was comparable to the concurrent control values throughout the entire study.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among hematological parameters were observed.
At the end of the administration period in females of test group 2 (300 mg/kg bw/d) platelet counts were significantly increased, but the alteration was not dose-dependent. Therefore, it was regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes among clinical chemistry parameters were observed.
At the end of the administration period in males of test groups 1 and 2 (100 and 300 mg/kg bw/d) glucose levels were significantly decreased, but the change was not dose-dependent. Therefore, it was regarded as incidental and not treatment-related.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Home cage observations
No test substance-related or spontaneous findings were observed in male and female animals of all test groups during the home cage observation.

Open field observations
The open field observations did not reveal any test substance-related findings in male and female animals of all test groups.

Sensorimotor tests/reflexes
There were no test substance-related findings in male and female animals of all test groups. One out of five examined female animals of dose group 1 showed vocalizations always when touched. Since this was not related to dose, it was assessed as spontaneous.

Quantitative Parameters
No test substance-related impaired parameters were observed in male and female animals of all test groups. The grip strength of forelimbs in females of dose group 3 was statistically significantly above the concurrent control values (8.0 versus [vs.] 5.1 in control). However, the mean value of the high-dose females was well within the range of the historical control data (HCD, GS F, mean value, range: 4.2 – 12.1) and was, therefore, assessed as incidental, not treatment-related and not adverse.

Motor activity measurement
No treatment-related, adverse changes on motor activity data (summation of all intervals) was observed in the male and female animals of all test substance-treated groups in comparison to the concurrent control values. The mean number of beam interrupts of the high-dose parental females was statistically significantly below the concurrent control values during interval 5. The numbers of beam interrupts of the intervals 1, 2 and 8 of the high-dose females were, however, above the control values. The decreased mean value of the high-dose females during one single interval was most likely an outlier since the summary value of intervals 1- 12 of the high-females was comparable to the control value. Therefore, the isolated decrease in interval 5 was assessed as incidental and not-treatment related. The statistically significantly increased number of beam interrupts in the low-dose females during interval 11 was considered to be spontaneous in nature and not treatment related since there was no relation to dose.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. This includes the 2 macroscopically detected foci in the glandular stomach of test group 2 females which correlated histopathologically to erosion/ulcer.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

In parental males (test groups 1, 2 and 3; 100, 300 and 1000 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycle data, generated during the last 2 weeks prior to mating for the F1 litter, revealed regular cycles in the females of all test groups 0 - 3. The mean estrous cycle duration was similar: 3.9 / 3.9 / 3.8 and 3.9 days in test groups 0 - 3, respectively.
In high dose females the different stages of functional bodies in the ovaries were present and comparable to the control animals.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

The stages of spermatogenesis in the testes of males of the high dose test group were comparable to those of the controls.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all test groups (0 - 3). Fertility was proven for all F0 parental males within the scheduled mating interval for F1 litter. Thus, the male fertility index was 100% in all test groups.
The female mating index calculated after the mating period for F1 litter was 100% in all test groups (0 - 3).
The mean duration until sperm was detected (GD 0) varied between 1.5 and 3.1 days without any relation to dosing.
All female rats delivered pups or had implants in utero: The fertility index was 100% in all test groups.
The mean duration of gestation values varied between 21.9 (control), 22.6** ([**p<=0.01] test group 1), 22.0 (test group 2) and 22.2 (test group 3). The statistically significantly increased number of gestation days in the low-dose females showed no relation to dose and was considered to be spontaneous in nature and not treatment related.
The gestation index was 100% in the control and test groups 2 - 3 and 90% in test group 1.
Implantation was not affected by the treatment since the mean number of implantation sites was comparable between all test substance-treated groups and the control, taking normal biological variation into account (12.1 / 11.0 / 11.4 and 12.3 implants/dam in test groups 0 - 3, respectively). Furthermore, there were no indications for test substance-induced intrauterine embryo-/fetolethality since the postimplantation loss did not show any significant differences between the groups, and the mean number of F1 pups delivered per dam remained unaffected (11.6 / 11.6 / 11.1 and 11.3 pups/dam in test groups 0 - 3, respectively)
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 99.1% / 98.1% / 98.2% and 100% in test groups 0 - 3, respectively. Moreover, the number of stillborn pups was not significantly different between the test groups.
Thus, the test substance Reaction mass of 2-methylbutyl acetate and pentyl acetate did not adversely affect reproduction and delivery of the F0 generation parental females.

Fertility
All female animals were pregnant.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test substance-related adverse clinical signs observed in any of the F1 generation pups of the different test groups.
One high-dose male pup (No. 8 of dam No. 132) had a thread-like tail during PND 0 - 4. This observation was not considered to be associated with the test compound since only one single pup was affected.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The mean number of delivered F1 pups per dam and the rates of liveborn, stillborn, found dead and cannibalized F1 pups were evenly distributed among the test groups. The respective values reflect the normal range of biological variation inherent in the strain used in this study.
The viability index indicating pup survival during lactation (PND 0 - 4) varied between 100%/ 98.2% / 99.2% and 100% in test groups 0 - 3, respectively.
The pups surviving index indicating pup survival during lactation (PND 4 - 13) was 100% in all test groups. Thus, the test substance did not influence pup survival in any of the treated groups (100, 300 and 1000 mg/kg bw/d).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weights and body weight change of all male and female pups in all test
substance-treated groups were comparable to the concurrent control values throughout the
entire study.
Three male runts were seen in the control, one male runt was seen in test group 1 and one
female runt was seen in test group 2.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

The anogenital distance and anogenital index of all test substance treated male and female pups was comparable to the concurrent control values.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A few pups showed spontaneous findings at gross necropsy, discolored testis, thread-like tail, dilated renal pelvis, dilated ureter, hydroureter and hydronephrosis.
These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences. Thus, all these findings were not considered to be associated to the test substance.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Sex ratio
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 13 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature.
Thyroid hormones
In female pups at PND 13 (test groups 11, 12 and 13; 100, 300 and 1000 mg/kg bw/d), no treatment-related alterations of T4 and TSH levels were observed. On PND 13 in male pups of test group 12 (300 mg/kg bw/d) T4 values were significantly increased, but the change was not dose-dependent. TSH values among these individuals were not altered as well as T4 and TSH values in test groups 11 and 13 (100 and 1000 mg/kg bw/d). Therefore, this T4 change in PND 13 male pups of test group 12 was regarded as incidental and not treatment-related.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Reproductive effects observed:

no

Conclusions:

In conclusion, under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats the oral administration of Reaction mass of 2-methylbutyl acetate and pentyl acetate by gavage to male and female Wistar rats resulted in no signs of systemic toxicity up to limit dose of 1000 mg/kg bw/d. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was the highest tested dose of 1000 mg/kg bw/d for male and female Wistar rats. The NOAEL for reproductive performance and fertility was set to 1000 mg/kg bw/d for male and female Wistar rats. The NOAEL for developmental toxicity was 1000 mg/kg bw/d.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Species:

rat

Quality of whole database:

Guideline study under GLP

Additional information

In an OECD guideline 422 compliant study, the test substance Reaction mass of 2-methylbutyl acetate and pentyl acetate was administered daily to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d, test groups 1-3, respectively) to screen for potential systemic, reproductive and developmental toxicity. The duration of treatment covered a 30 days in-life period in males (including premating, mating [mating pairs were from the same test group] and postmating period) and a 2-weeks premating and mating period, the entire gestation and approximately 3 weeks of lactation period in females. Parental females were allowed to give birth and bring up the offspring until sacrifice on PND 4 or 13.

Regarding clinical examination, no test substance related, adverse signs of (systemic) toxicity were observed up to limit dose of 1000 mg/ kg bw/d. All males and females of the high-dose (1000 mg/kg bw/d) and two out of ten males of the middose (300 mg/kg bw/d) showed salivation immediately after dosing (up to 2 hours post dosing) during the treatment period. It is most likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse toxicologically relevant finding. Neither water and food consumption nor body weight were adversely affected in any of the tested dose groups. Regarding clinical pathology, no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d. Regarding pathology, there were no treatment-related findings in organ weights, gross or microscopic pathology. All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment. Regarding fertility and reproductive performance, no signs of toxicity were observed in male or female parental animals of all test groups during the entire study. F0 parental animals proved to be fertile. Mating behavior, conception, implantation and parturition were not affected. Regarding developmental toxicity, no signs of developmental toxicity were noted in any of the treated groups. Pup status, viability, survival and growth showed no treatment-related, adverse findings. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

In conclusion, under the conditions of the present OECD 422 combined repeated dose toxicity study with the reproductive/developmental screening test in Wistar rats the oral administration of Reaction mass of 2-methylbutyl acetate and pentyl acetate by gavage to male and female Wistar rats resulted in no signs of systemic toxicity up to limit dose of 1000 mg/kg bw/d. Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was the highest tested dose of 1000 mg/kg bw/d for male and female Wistar rats. The NOAEL for reproductive performance and fertility was set to 1000 mg/kg bw/d for male and female Wistar rats. The NOAEL for developmental toxicity was 1000 mg/kg bw/d.

In a subsequent OECD guideline 443 compliant study, reaction mass of 2 -methylbutyl acetate and pentyl acetate was administered to groups of 25 male and 25 female healthy young Wistar rats as test groups 00 - 03 as an aqueous preparation by stomach tube at different dosages (0, 100, 300 and 1000 mg/kg body weight/day [mg/kgbw/d]). F0 animals were treated at least for 10 weeks prior to mating to produce a litter (F1 generation). Mating pairs were from the same dose group. Pups of the F1 litter were selected (F1 rearing animals) and assigned to 2 different cohorts (1A, 1B) which were subjected to specific postweaning examinations. The study was terminated with the terminal sacrifice of the F1 rearing animals of cohort 1A. Control animals were dosed daily with the vehicle (0.5% CMC suspension in deionized water with Cremophor EL [10 mg/100mL]).

In general, analyses confirmed the prepared concentrations, the homogeneous distribution and the stability of the test substance in the vehicle.

There were no test substance-related mortalities or adverse clinical observations,indicating systemic toxicity, noted in any of the groups. In particular,regularly conducted detailed clinical observations revealed no test substance-related adverse effects.Transient salivation during a short time period after gavage dosing was noted for nearly all high-dose animals during all sections of the study. It is likely, that this temporary finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. It is, however, not considered to be an adverse finding indicating systemic toxicity.

Food consumption was essentially comparable to the concurrent control values in all F0, F1A and F1B dose groups throughout the study. Some minor and patchy changes which followed no dose-response were considered to be chance findings rather than associated with the treatment.

Body weights and body weight change of all test substance-treated male and female F0, F1A and F1 Brats were essentially comparable to the concurrent control values throughout the entire study. Some minor and patchy changes which followed no dose-response were considered to be chance findings rather than associated with thetreatment.

Concerning clinical pathology, in F0 as well as F1 rats no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d. Regarding pathology, no test substance-related adverse findings were noted in any of the dose groups.

F0 GENERATION PARENTAL ANIMALS: All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

 The reproductive organs of the mating pair suspected of reduced fertility did not show histopathological findings that could explain the reduced fertility.

F1 GENERATION, REARING ANIMALS, COHORT 1A: All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation totreatment.

The results of the differential ovarian follicle count (DOFC) – comprising the numbers of primordial and growing follicles, as well as the combined incidence of primordial plus growing follicles–showed no significant differences between the placebo group 10 and animals of test group13.

 F1 GENERATION, REARING ANIMALS, COHORT 1B: One animal of this cohort died. Beside of dilated lungs, no gross finding was noted that could explain the death in this animal. 

SURPLUS F1 GENERATION PUPS ON PND 22 (F1 WEANLINGS NOT SELECTED FOR COHORTS): All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

There were no indications from clinical examinations as well as gross and histopathology, that Reaction mass of2 -methylbutyl acetate and pentyl acetate adversely affected the fertility or reproductive performance of the F0 parental animals up to and including the administered high-dose of 1000 mg/kg bw/d. Estrous cycle data, sperm quality of males, mating behavior, conception, gestation, parturition, lactation and weaning as well as sexual organ weights and gross and histopathological findings of these organs (specifically the differential ovarian follicle count)were comparable between the rats of all groups including control and ranged within the historical control data of the test facility.

For all liveborn male and female pups of the F0 parents, no test substance-induced signs of developmental toxicity were noted at dose levels as high as 1000 mg/kg bw/d. Postnatal survival, pup body weight gain as well as post-weaning development of the offspring of this test group until puberty remained unaffected by the test substance. Furthermore, clinical and/or gross necropsy examinations of the F1 pups revealed no adverse findings.

Measurement of thyroid hormones revealed no effect caused by the test substance, neither in the F0 parental animals nor in the liveborn F1 offspring.

Neither the anogenital distance/index nor the check for the presence of nipples/areolas, both very sensitive marker of potential endocrine-mediated imbalances, revealed any test substance-related effects.

Vaginal opening and preputial separation are commonly used developmental markers for onset of puberty in laboratory rats. No delays beyond a normal range of biological variation in rat (multi)generation studies which might be attributable to the treatment were noted in any of the test substance-treated groups.

Thus, under the conditions of the present extended one-generation reproduction toxicity study the NOAEL (no observed adverse effect level) for general, systemic toxicity is 1000 mg/kg bw/d, the highest tested dose.

The NOAEL for fertility and reproductive performance for the parental rats is 1000 mg/kg bw/d, the highest tested dose.

The NOAEL for developmental toxicity in the F1 progeny is 1000 mg/kg bw/d, the highest tested dose.

Effects on developmental toxicity

Description of key information

OECD 414, rat: no malformations (dev. NOAEC = 1500 ppm)

OECD 414, rabbit: no malformations (dev. NOAEC = 1500 ppm)

(UCC 1994)

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 Oct 1990 - 16 Nov 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Study was conducted prior to actual guidelines but followed intent of the guidelines

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Seventy (70) virgin female New Zealand White rabbits (3.0-3.5 kg, as designated by the supplier, and approximately 5 months old) were received on
October 1, 1990 from Hazleton Dutchland Laboratories, Inc. (Denver, PA). Male New Zealand White rabbits from the BRRC buck colony (originally from the same supplier) were used for breeding.

A pretest health screen, utilizing three females randomly selected from the population of rabbits received for the study, was performed within 1 day of receipt of the animals. Examination for intestinal parasites was performed by the zinc sulfate flotation method. Evaluation for fecal parasites indicated that the animals were free of parasites and considered to be acceptable for use on the study.

The females were individually housed in stainless steel, wire-mesh cages, 61 cm x 61 cm x 41 cm, during the acclimation and study periods. A layer of Deotized Animal Cage Boarde (Shepherd Specialty Papers, Inc., Kalamazoo, MI) was placed under each row of cages to collect solid and liquid excrement. The paper board was changed at least three times per week. Other animal care was performed regularly according to BRRC standard operating procedure. The room temperature and relative humidity were monitored continuously by a Cole-Parmer Hygrothermographo Seven-Day Continuous Recorder, Model No. 8368-00 (Cole-Parmer Instrument Co., Chicago, IL). Temperature and relative humidity were routinely maintained within the protocol-specified range of 61-70°F and 40-70%, respectively. Any minor exceptions to these specified ranges were noted in the raw data. The animals were kept on a 12-hour photoperiod using an automatic timer throughout the study.

During nonexposure periods, water and food were available to the animals ad libitum. Tap water (Municipal Authority of Westmoreland County, Greensburg, PA) was delivered by an automatic watering system with demand control valves mounted on each rack. Water analyses were provided by the supplier, the Halliburton NUS Environmental Laboratories, Materials Engineering & Testing Company, and Lancaster Laboratories, Inc. at regular intervals. EPA standards for maximum levels of contaminants were not exceeded. The animals were supplied with Agway Prolab Certified Rabbit Foodo, Batch No. AUGUST 15 90 W1 (Agway, Inc., St. Marys, OH). Analyses for chemical composition and possible contaminants of each feed lot were performed by Agway, Inc. (Ithaca, NY), and the results were included in the raw data.

During the exposures, the animals were individually housed, separated by exposure group, in stainless steel, wire-mesh cages, 35 cm x 37 cm x 41 cm. Food and water were withheld during the exposures.

The body weight and physical condition of all animals were monitored for approximately 2 weeks prior to mating.

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

The inhalation chambers used for this study were constructed from stainless steel with glass windows (Wahmann Manufacturing Company, Timonium, MD) for animal observation. The volume of each chamber was approximately 4320 liters, and the air flow was 1000 liters/minute (14 air changes per hour). Chamber temperature and relative humidity were recorded using Industrial thermometers (Control Specialties, Inc., Houston, TX) and an Airguide humidity indicator (Airguide Instrument Co., Chicago, IL). Temperature and relative humidity measurements were recorded twelve times per exposure. Prior to the start of the study, the distribution of primary amyl acetate vapor in the chamber was evaluated at five different locations in two of the three exposure chambers. In addition, prior to the start of exposures, and on Exposure Days 2 and 16, the oxygen content of all chambers was measured with an 02 Indicator Model 250 (Mine Safety Appliances, Pittsburgh, PA).

Primary Amy1 Acetate Vapor Generation
Liquid primary amyl acetate was metered from a piston pump (Fluid Metering, Inc., Oyster Bay, NY) into a heated glass evaporator similar in design to that described by Carpenter et al. (1975) and, more recently, by Snellings and Dodd (1990). The temperature in the evaporator(s) was maintained at the lowest level sufficient to vaporize the test substance. The resultant vapor was carried into the chamber by a countercurrent air stream that entered the bottom of the evaporator. Prior to and after the exposure regimen, temperature was measured from the inside surface of the evaporators using a Model 400A Doric Trendicator (Doric Scientific Division, San Diego, CA) and a Type K thermocouple.

Exposure Regimen
The mated females assigned to the study were exposed to primary amyl acetate vapor or filtered air for six hours per day during the period of major
organogenesis (gd 6 through 18, October 21 through November 5, 1990). The 6-hour exposure interval was defined as the time when the vapor generation system was turned on and subsequently turned off. Within each chamber, the cage placement pattern was changed daily to compensate for any possible, but undetected, variations in chamber exposure conditions.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber concentrations of primary amyl acetate vapor were analyzed by flame ionization gas chromatography. Approximately nine samples were obtained from each exposure chamber including the control chamber during the 6-hour exposure period. The nominal concentration was calculated from the total amount of test substance delivered to the chamber and chamber air flow rate.

Details on mating procedure:

New Zealand White rabbit females were mated to "provenn males from the BRRC breeding colony. Following successful copulation, the animals were returned to their respective cages; the date of the copulation was designated gestation day (gd) 0. The mating period was October 15-18, 1990. On each mating day successfully mated females were assigned to one of three exposure groups or one air-exposed control group using a computer-based randomization program. At completion of mating, each exposure group consisted of 15 mated female rabbits.

Duration of treatment / exposure:

6-hour exposure on gestation days 6-18

Frequency of treatment:

Daily on gestation days 6-18

Duration of test:

gestation days 6-18

Remarks:

Doses / Concentrations:
0, 500, 1000 and 1500 ppm
Basis:
other: target concentration

Remarks:

Doses / Concentrations:
0, 509±4.6, 995±9.2, and 1522±20.1 ppm (corresponding to approx. 2.7, 5.4 and 8.1 mg/L air)
Basis:
analytical conc.

No. of animals per sex per dose:

15 timed-pregnant New Zealand White rabbits

Control animals:

yes, concurrent no treatment

Details on study design:

Target Concentration Selection
The developmental toxicity range-finding study was conducted at target concentrations of 0, 500, 1000, and 1500 ppm primary amyl acetate vapor. Selection of target vapor concentrations for the range-finding study was based on a previous 9-day vapor inhalation study in rats and other available acute toxicity data (see BRRC Report 47-103). Following acute dynamic exposure of Wistar rats to concentrations up to 5.2 mg/L, decreases in body weight gain were observed on postexposure day 1, but weight gains returned to normal limits by postexposure day 3. No deaths occurred following 6-hour static exposures of male and female Wistar rats to concentrations up to 19.3 and 19.7 mg/L primary amyl acetate vapor, respectively. In the 9-day study, repeated exposure of Wistar rats to concentrations up to 1200 ppm produced no clear evidence of toxicity. Although above the limit test exposure level of 5 mg/L (940 ppm), 1500 ppm was selected as the high vapor concentration for the developmental toxicity exposure range-finding study in order to maximize the likelihood of producing sustained maternal effects. While no concentration-related maternal effects were observed in the range-finding study, target concentrations of 0 (control), 500, 1000, and 1500 ppm primary amyl acetate vapor (the same as those in the range-finding study) were selected for the definitive study by the Sponsor. A high vapor concentration of 1500 ppm was selected in order to maximize the likelihood of producing sustained maternal effects using a larger number of animals in the dose group.

Maternal examinations:

Maternal In-Life Observations and Evaluations
All does were observed twice daily for morbidity and mortality. Does were observed for clinical signs of toxicity once daily. In addition, during the
exposures, animals were observed from outside their respective exposure chambers for overt signs. Maternal body weights were taken on gd 0, 6, 12, 18, 24, and 29. Food consumption was measured daily throughout the study (gd 0 through 29).

Ovaries and uterine content:

Maternal Necropsy and Laparotomy
At scheduled sacrifice on gd 29, does were sacrificed by injection of Beuthanasia-D Special solution (Schering Corporation USA, Kenilworth, NJ).
The maternal sacrifice period was November 13-16, 1990.

The maternal body cavities were opened by midline thoracolaparotomy. The uterus, ovaries ( including corpora lutea) , cervix, vagina, and abdominal and thoracic cavities were examined grossly. Ovarian corpora lutea of pregnancy were counted. The maternal liver and kidneys were weighed. Each uterus was externally examined for signs of hemorrhage, removed from the peritoneal cavity, weighed and dissected longitudinally to expose the contents. All live and dead fetuses and resorption sites (early and late) were noted and recorded. Uteri from females that appeared nongravid were placed in a 10% ammonium sulfide solution for detection of early resorptions (Salewski, 1964).

Reference
Salewski, E. (1964). Farbemethode Zum Makroskopischen Nachweis Von Implantations-Stellen am Uterus Der Ratte. Naunyn-Schmiedebergs, Arch.
Exp. Pathol. Pharmakol. 247, 367.

Fetal examinations:

Immediately upon exteriorization, fetuses were euthanized by intraperitoneal injection of sodium pentobarbital. All live and dead fetuses were weighed and examined externally for variations and malformations including cleft palate. All live fetuses in each litter were examined for thoracic and abdominal visceral abnormalities by methods described by Staples (1974). Fetal sexes were determined during the visceral examination. One-half of the live fetuses (even-numbered fetuses from litters with an even number of live fetuses, odd numbered fetuses from litters with an odd number of live fetuses) in each litter were decapitated and their heads were fixed in Bouin's solution for examination of craniofacial structures by sectioning methods modified from van Julsingha and Bennett (1977). All fetuses (50% intact, 50% decapitated) in each litter were eviscerated, air-dried, and processed for skeletal staining with alizarin red S (Crary, 1962; Peltzer and Schardein, 1966), and examined for skeletal malformations and variations.

References
Crary, D. D. (1962). Modified Benzyl Alcohol Clearing of Alizarin-Stained Specimens without Loss of Flexibility. Staining Technology 37, 124-125.

Peltzer, M. A. and Schardein, J. L. (1966). A convenient method for processing fetuses for skeletal staining. Stain Technology 4l, 300-302.

Staples, R. E. (1974). Detection of visceral alterations in mammalian fetuses. Teratology, 9, A-37.

van Julsingha, E. B. and C. G. Bennett (1977). A dissecting Procedure for the Detection of Anomalies in the Rabbit Foetal Head. In: Methods in Prenatal Toxicology (D. Neubert, H. J. Merker, and T. E. Kwasigroch, Editors). PsG Publishing Company, Inc., Littleton, Massachusetts, pp. 126-144.

Statistics:

The unit of comparison was the pregnant dam or the litter. The data for quantitative continuous variables were intercompared for the exposure groups and the control group by use of Levene's test for equality of variances, analysis of variance (ANOVA), and t-tests. The t-tests were used when the F value from the ANOVA was significant. When Levene's test indicated similar variances, and the ANOVA was significant, a pooled t-test was used for pairwise comparisons. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances followed, when necessary, by a separate variance t-test for pairwise comparisons.

Nonparametric data obtained following laparohysterectomy were statistically evaluated using the Kruskal-Wallis test followed by the Mann-Whitney U-test when appropriate. Incidence data were compared using Fisher's Exact Test. With the exception of the data analysis for fetal malformations and variations, all statistical analyses were performed using BMDP Statistical Software (Dixon, 1990). Incidence data for fetal malformations and variations were analyzed using a program developed at BRRC. For all statistical tests, the probability value of < 0.05 (two-tailed) was used as the critical level of significance.

Indices:

No data

Historical control data:

No additional information available.

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: Effects at 1500 ppm included body weight losses during the first week of the exposure period and reduced food consumption during the entire exposure period.

Details on maternal toxic effects:
Chamber Concentration, Temperature, and Relative Humidity
The means of daily mean analytical concentrations (+/- SD) were 509 (+/- 4.6), 995 (+/-9.2), and 1522 (+/- 20.1) ppm for target concentrations of 500, 1000, and 1500 ppm, respectively. The overall mean A/N ratios were 1.03, 1.02, and 1.05 for the 500, 1000, and 1500 ppm concentration, respectively. Among exposure groups, the daily mean chamber temperature and relative humidity ranged from 21-23C and 45-50%, respectively. For all measurements, the chamber oxygen content was 20.8%.

Animal Fate and Observations
No mortality was observed in any exposure group prior to scheduled sacrifice. One doe in the 500 ppm group aborted on gd 24. Two females each from the control and 1500 ppm exposure groups were not pregnant. All pregnant females contained viable fetuses. The overall pregnancy rate was equivalent for all exposure groups, ranging from 87 to 100%. Exposure-related clinical signs observed during the study included full food hoppers for several does in the 1500 ppm group.

Maternal Food Consumption
Food consumption was generally decreased in the 1500 ppm group during the exposure period with differences attaining statistical significance on gd 7-8, 8-9, 9-10, and 11-12. The statistically significant decrease observed in the 1000 ppm group for gd 7-8 was not considered to be biologically significant due to the transient nature of the decrease and the fact that the decrease was observed on the day after the first exposure.

Maternal Body Weights and Weight Changes
No statistically significant differences in body weights were observed in any exposure group. Statistically significant body weight losses were observed in the 1000 and 1500 ppm groups during the gd 6 to 12 interval. In addition, body weight loss was observed in the 500 ppm group. The body weight losses observed in the 1500 ppm group were considered to be related to the exposure since during this period, all does in this group except one lost weight. This body weight loss early in the exposure period contributed to the slightly reduced body weight gain observed during the entire exposure period. Losses observed in the 500 and 1000 ppm groups were not considered to be biologically significant since the magnitude of the losses was small, were within the range of losses commonly seen in rabbit studies, and were unaccompanied by consistent reductions in food consumption.

Maternal Necropsy and Laparotomy
There were no exposure-related necropsy findings in the does at scheduled sacrifice on gd 29 or in the doe which aborted prior to scheduled sacrifice. There were no statistically significant differences in terminal body weight, liver weight or kidney weight. While not statistically significant, corrected weight changes appeared to be decreased in the 1500 ppm group. There were no effects of exposure on the number of ovarian corpora lutea, on the number of total, viable or nonviable (early and late resorptions and dead fetuses) implantations per litter or on sex ratio (percent males). Percent preimplantation and postimplantation losses were equivalent across groups.

Dose descriptor:

NOEC

Effect level:

5.4 mg/L air (analytical)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no effects of exposure on the number of ovarian corpora lutea, on the number of total, viable or nonviable (early and late resorptions and dead fetuses) implantations per litter or on sex ratio (percent males). Percent preimplantation and postimplantation losses were equivalent across groups.

No exposure-related effects on fetal body weights (all fetuses, male or female) were observed in any group. Statistically significant decreases
observed in mean fetal weight of male or female fetuses in the 1000 ppm group were not considered to be exposure-related due to the lack of concentration response relationship.

There were no statistically significant differences in individual external, visceral or skeletal malformations by category, or of total malformations
among all groups. There were no statistically significant differences among groups in the incidence of individual fetal external or visceral variations. Of the 73 different types of individual fetal skeletal variations observed, none were statistically significantly increased due to exposure. There were no exposure-related increases in the incidence of variations by category (external, visceral or skeletal) or of total variations.

Dose descriptor:

NOAEC

Effect level:

>= 8.1 mg/L air (analytical)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

no effects observed

Developmental effects observed:

no

Conclusions:

Exposure of pregnant New Zealand White rabbits to primary amyl acetate vapor during organogenesis at 0, 500, 1000, and 1500 ppm resulted in minimal maternal toxicity at 1500 ppm and no embryolethality or developmental toxicity (including teratogenicity). The no-observed-effect level" (NOEL) for maternal toxicity was 1000 ppm. The NOEL for developmental toxicity was greater than 1500 ppm.

Executive summary:

Four groups, each consisting of 15 timed-pregnant New Zealand White rabbits were exposed to primary amyl acetate vapor for six hours/day on gestational days (gd) 6 through 18. Target concentrations of primary amyl acetate vapor were 0 (control), 500, 1000, and 1500 ppm. Clinical observations were made daily, and body weights were measured on gd 0, 6, 12, 18, 24, and 29. Maternal food consumption was measured daily throughout gestation, gd 0-29. At scheduled sacrifice on gd 29, the does were subjected to a necropsy and were evaluated for body weight, liver and kidney weights, and gravid uterine weight. Ovarian corpora lutea of pregnancy were counted and status of implantation sites (i.e. resorptions, dead fetuses, live fetuses) were identified and recorded. All fetuses were dissected from the uterus, counted, weighed, and examined for external abnormalities. All live fetuses in each litter were examined for visceral malformations and variations and sexed. Approximately one-half of the live fetuses in each litter were examined for craniofacial malformations and variations. All fetuses (50% intact, 50% decapitated) in each litter were stained with alizarin red S and examined for skeletal malformations and variations.

No mortality occurred during the study. One female in the 500 ppm group aborted. The pregnancy rate was equivalent for all groups and ranged from 87 to 1001. Maternal toxicity was observed in the 1500 ppm group and included body weight losses during the first six days of the exposure period accompanied by reduced food consumption during the entire exposure period.

Fetal examination indicated no evidence of fetotoxicity or developmental toxicity in any of the exposure groups. The number of total, viable or nonviable implantations per litter, the sex ratio, and the percent preimplantation and postimplantation losses were similar in all groups. No clear exposure-related decreases in mean fetal body weight were observed. External, visceral and skeletal examinations of the fetuses revealed no exposure-related differences in the incidences of variations or malformations. Exposure of pregnant New Zealand White rabbits during organogenesis to primary amyl acetate vapor at 0, 500, 1000, and 1500 ppm resulted in maternal toxicity at 1500 ppm and no developmental toxicity (including teratogenicity). The no-observed-effect level (NOEL) for maternal toxicity was 1000 ppm. The NOEL for developmental toxicity was at least 1500 ppm.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Oct - 9 Nov 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Study was conducted prior to actual guidelines but followed intent of the guidelines

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Details on test animals or test system and environmental conditions:

One hundred sixty-two virgin male and 162 virgin female Fischer 344 [CDF F-344 Cr1:BRl rats were received from Charles River Breeding Laboratories, Inc., Kingston, NY, on October 1, 1990. The date of birth was July 23, 1990 for the males and July 30, 1990 for the females. Males were 70 days old upon arrival and weighed approximately 190-215 g; females were 63 days old and weighed approximately 135-147 g.

Within two days of receipt, the animals were examined by a Clinical Veterinarian and representative animals were subjected to a pretest health screen including full necropsy, examination for intestinal parasites, histologic examination of selected tissues, and serum viral antibody analyses. Based on the results of these data, the Clinical Veterinarian indicated that these animals were in good health and suitable for use.

The females were housed two to a cage in stainless steel, wire-mesh cages (30.5 em x 15.5 cm x 18 cm) during the acclimation period. Females placed on study were singly housed in stainless steel wire-mesh caging (22.5 x 15.5 x 18.0 cm), except during the mating period. During the mating period, animals were housed in stainless steel, wire-mesh cages, 22.5 cm x 31.0 cm x 18 cm. During the exposures, the animals were individually housed. Each exposure group was housed in a separate carrier in stainless steel, wire-mesh cages, 21 cm x 12.5 cm x 18 cm. The animals were kept in Room 164 from receipt to October 10, 1990, and in Room 106 from October 10, 1990 until completion of the study. A layer of Deotized Animal Cage Board (Shepherd Specialty Papers, Inc., Kalamazoo, MI) was placed under each row of cages to collect solid and liquid excrement. The paperboard was changed at least three times per week. Other animal care was performed regularly according to BRRC standard operating procedure. Room temperature and relative humidity were monitored continuously using a Cole-Parmer Hygrothermograph Seven-Day Continuous Recorder, Model No. 8368-00 (Cole-Parmer Instrument Co., Chicago, IL). Temperature and relative humidity were routinely maintained at 66-77F and 40-70%, respectively. Throughout the study, the animals were kept on a 12-hour photoperiod using an automatic timer.

Tap water (Municipal Authority of Westmoreland County, Greensburg, PA) was available libitum except during exposures and was delivered by an automatic watering system with demand control valves mounted on each rack. Water analyses were provided by the supplier, the Halliburton NUS Environmental Laboratories, Materials Engineering & Testing Company, and Lancaster Laboratories, Inc. at regular intervals. EPA standards for maximum levels of contaminants were not exceeded. Agway certified Ground Rodent Chow (Batch No. MAY 17 90 W2; Agway, Inc., St. Marys, OH) was available ad libitum except during exposures. Analyses for chemical composition and possible contaminants of the feed lot used for the study were performed by Agway Inc. and the results were included in the raw data.

The body weight and physical condition of all animals were monitored for approximately 2 weeks prior to mating.

Twenty-five (25) plug-positive females were assigned to each experimental group by a randomization procedure based on stratification of gd 0 body weights such that all groups were equivalent in mean body weight and body weight range on gd 0. Mean body weights of females placed on study were 154.6-154.8 grams. The mating period was October 15-18, 1990; gd 0 was October 16-19, 1990.

Route of administration:

inhalation: vapour

Type of inhalation exposure (if applicable):

whole body

Vehicle:

unchanged (no vehicle)

Details on exposure:

The inhalation chambers used for this study were constructed from stainless steel with glass windows (Wahmann Manufacturing Company, Timonium, MD) for animal observation. Details of the chamber design are included in Appendices 1B and 1C. The volume of each chamber was approximately 4320 liters, and the air flow was 1000 liters/minute (14 air changes per hour). Chamber temperature and relative humidity were recorded using Industrial thermometers (Control Specialties, Inc., Houston, TX) and an Airguide humidity indicator (Airguide Instrument Co., Chicago, IL). Temperature and relative humidity measurements were recorded approximately every 30 minutes during each exposure. Prior to the start of exposures, chamber distribution of primary amyl acetate vapor in the chamber was evaluated at five different locations in two of the three exposure chambers (500 and 1500 ppm target concentrations). Details of the chamber distribution are included in Appendix 1C. In addition, prior to the start of exposures and on Exposure Days 2 and 16, the oxygen content of all chambers were measured with an 02 Indicator Model 250 (Mine Safety Appliances, Pittsburgh, PA).

Tarqet Concentrations
Target primary amyl acetate vapor concentrations of 0 (control), 500, 1000, and 1500 ppm were selected for this study by the Sponsor based on the results from an exposure range-finding study of primary amyl acetate vapor in pregnant Fischer 344 rats.

Primary Amyl Acetate Vapor Generation
Liquid primary amyl acetate was metered from a piston pump (Fluid Metering, Inc., Oyster Bay, NY) into a heated glass evaporator similar in design to that described by Carpenter et e. (1975) and, more recently, by Snellings and Dodd (1990). Prior to and at the end of the exposure regimen, temperature was measured from the inside surface of the evaporators using a Model 400A Doric Trendicator (Doric Scientific Division, San Diego, CA) and a Type K thermocouple. The temperature in the evaporator(s) was maintained at the lowest level sufficient to vaporize the test substance. The resultant vapor was carried into the chamber by a countercurrent air stream that entered the bottom of the evaporator.

References
Carpenter, C. P., E. R. Kinkead, D. L. Geary, Jr., L. J. Sullivan, and J. M. King (1975). Petroleum Hydrocarbon toxicity Studies. I. Methodology. Toxicol. Appl. Pharmacol. 32, 246-262.

Snellings, W. M. and Dodd, D. E. (1990). In Handbook of In Vivo Toxicity Testinq (D. L. Arnold, H. C. Grice, and D. R. Krewski, eds.) 186-246. Academic Press, New York.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber concentrations of primary amyl acetate vapor were analyzed by flame ionization gas chromatography. Approximately nine samples were obtained from each exposure chamber including the control chamber during the 6-hour exposure period. The nominal concentration was calculated from the total amount of test substance delivered to the chamber and chamber air flow rate.

Details on mating procedure:

Virgin female Fischer 344 rats were paired with virgin male Fischer 344 rats on a one male to one female basis. Each male was mated only once in this study. The observation of vaginal or dropped copulation plugs was considered evidence of successful mating. The day a copulation plug was found was designated gd 0. After successful mating, plug-positive females were housed individually.

Duration of treatment / exposure:

6 hrs/day on gestation days 6-15

Frequency of treatment:

Daily

Duration of test:

gestation days 6-15

Remarks:

Doses / Concentrations:
0, 500, 1000 and 1500 ppm
Basis:
other: target concentration

Remarks:

Doses / Concentrations:
0, 509±4.6, 995±9.2, and 1522±20.1 ppm (corresponding to approx. 0, 2.7, 5.4 and 8.1 mg/L air)
Basis:
analytical conc.

No. of animals per sex per dose:

25 timed-pregnant Fischer 344 rats/dose level

Control animals:

yes, concurrent no treatment

Details on study design:

In a probe study four groups, each consisting of 8 timed-pregnant Fischer 344 rats were exposed for 6 hours/day on gestational days (gd) 6 through 15 to primary amyl acetate vapor. Target concentrations of primary amyl acetate were 0 (control), 500, 1000, and 1500 ppm. Clinical observations were made daily and body weights were measured on gd 0 , 6, 9, 12, 15, 18 and 21. Maternal food consumption was measured at 3 day intervals throughout gestation, gd 0-21. At scheduled sacrifice on gd 21, the dams were evaluated for body weight, liver and kidney weights, gravid uterine weight and status of implantation sites (i.e., resorptions, dead fetuses, live fetuses). All fetuses in each litter were weighed, sexed and examined for external malformations and variations.

No mortality or frank maternal toxicity was observed during the study. Six females, two each from the control and 1000 ppm groups, and one each from the 500 and 1500 ppm groups, delivered early and were removed from the study. The overall pregnancy rate was equivalent among groups and ranged from 75 to 87.5%.

Slightly decreased body weight gain and food consumption were observed during gd 6-9 in the 1000 and 1500 ppm groups, The number of total and nonviable implantations per litter and the percent preimplantation losses were similar in all groups. The number of viable implants were increased in all primary amyl acetate exposure groups. No exposure-related decreases in the mean fetal body weight per litter were observed. External examination of the fetuses revealed no differences in the incidences among groups.

Based on the results of this study, exposure levels of 500, 1000 or 1500 ppm primary amyl acetate were selected for a subsequent definitive teratology study in Fischer 344 rats.

Females assigned to the study were exposed to primary amyl acetate vapor or filtered air for six hours per day on gd 6 through 15. The 6-hour exposure interval was defined as the time when the vapor generation system was turned on and subsequently turned off. Within each chamber, the cage placement pattern was changed daily to compensate for any possible, but undetected, variations in chamber exposure conditions. A description of cage rotation within exposure chambers can be found in the study notebook. The exposure period was October 22 through November 3, 1990.

Maternal examinations:

Maternal In-Life Observations and Evaluations
All dams were observed twice daily for morbidity and mortality. Dams were observed for clinical signs of toxicity once daily. In addition, during the actual exposures, animals were observed from outside their respective exposure chambers for overt signs. Maternal body weights were taken on gd 0, 6, 9, 12, 15, 18 and 21. Food consumption was measured at 3-day intervals throughout the study (gd 0 through 21).

Ovaries and uterine content:

At scheduled sacrifice on gd 21, dams were sacrificed by carbon dioxide asphyxiation. The maternal sacrifice period was November 6-9, 1990.

The maternal body cavities were opened by midline thoracolaparotomy. The uterus, ovaries (including corpora lutes), cervix, vagina, and peritoneal and thoracic cavities were examined grossly. Ovarian corpora lutea of pregnancy were counted. The maternal liver and kidneys were weighed. Each uterus was externally examined for signs of hemorrhage, removed from the peritoneal cavity, weighed and dissected longitudinally to expose the contents. All live and dead fetuses and resorption sites (early and late) were noted and recorded. Uteri from females that appeared nongravid were placed in a 10% ammonium sulfide solution for detection of early resorptions (Salewski, 1964).

Reference
Salewski, E. (1964). Farbemethode Zum Makroskopischen Nachweis Von Implantations-Stellen am Uterus Der Ratte. Naunyn-Schmiedebergs, Arch. Exp. Pathol. Pharmakol. 247, 367

Fetal examinations:

All live fetuses were weighed, sexed, and examined for external variations and malformations including cleft palate. Approximately one-half of the live
fetuses (even-numbered fetuses from litters with an even number of live fetuses, odd-numbered fetuses from litters with an odd number of live fetuses) in each litter were examined for thoracic and abdominal visceral abnormalities by a modification of methods described by Staples (1974). These fetuses were then decapitated and their heads were fixed in Bouin's solution for examination of craniofacial structures by sectioning methods modified from Wilson (1965; 1973). The remaining half of the fetuses (intact) in each litter were eviscerated, fixed in ethanol, processed for skeletal staining with alizarin red Sf and examined for skeletal malformations and variations.

References
Staples, R. E. (1974). Detection of visceral alterations in mammalian fetuses. Teratology, 9, A-37.

Wilson, J. G. (1965). Embryological considerations in teratology. In: Teratoloqy Principles and Techniques (J. G. Wilson and J. Warkany, Editors). The University of Chicago Press, pp. 251-277.

Wilson, J. G. (1973). Environment and Birth Defects. Academic Press, NY,

Statistics:

The unit of comparison was the pregnant dam or the litter. The data for quantitative continuous variables were intercompared for the exposure groups and the control group by use of Levene's test for equality of variances, analysis of variance (ANOVA), and t-tests. The t-tests were used when the F value from the ANOVA was significant. When Levene's test indicated similar variances, and the ANOVA was significant, a pooled t-test was used for pairwise comparisons. When Levene's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances followed, when necessary, by a separate variance t-test for pairwise comparisons.

Nonparametric data obtained following laparohysterectomy were statistically evaluated using the Kruskal-Wallis test followed by the Mann-Whitney U-test when appropriate. Incidence data were compared using Fisher's Exact Test. With the exception of the data analysis for fetal malformations and variations, all statistical analyses were performed using BMDP Statistical Software (Dixon, 1990). Incidence data for fetal malformations and variations were analyzed using a program developed at BRRC. For all statistical tests, the probability value of < 0.05 (two-tailed) was used as the critical level of significance.

References
Dixon, W. J. (1990). BMDP Statistical Software. University of California Press, Berkeley, CA.

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: Statistically significant decreases in corrected body weight gain were observed in the 1500 and 1000 ppm groups and a slight decrease was observed in the 500 ppm group.

Details on maternal toxic effects:
Chamber Concentration, Temperature, and Relative Humidity
The means of daily mean analytical concentrations (+/- SD) were 509 (+/- 4.6), 995 (+/- 9.2), and 1522 (+/- 20.1) ppm for target concentrations of 500, 1000 and 1500 ppm, respectively. The overall mean A/N ratios were 1.03, 1.02 and 1.05 for the 500, 1000 and 1500 ppm concentration, respectively. Among exposure groups, the daily mean chamber temperature and relative humidity ranged from 21-23C and 45-50%, respectively. For all measurements, the chamber oxygen content was 20.8%.

Animal Fate
No females aborted, delivered early or were removed during the study. One female each from the control and all primary amyl acetate exposure groups was not pregnant. All pregnant females contained viable fetuses. The overall pregnancy rate was the same for all groups, 96%. All subsequent presentations and discussions of summary data are based on pregnant animals.

Clinical Observations
No exposure-related clinical signs were observed during the study in any exposure group.

Maternal Body Weiqhts and Weight Changes
No statistically significant differences in body weights were observed in any exposure group. Although not statistically significant, body weight gains were decreased during the first three days of the exposure period in the 1500 ppm group. No other effects on body weight gains were observed.

Maternal Food Consumption
No statistically significant differences in food consumption were observed in any exposure group during the preexposure and exposure periods. Statistically significant decreases in food consumption were observed in all primary amyl acetate-exposed groups during the postexposure period.

Maternal Necropsy and Laparotomy
There were no exposure-related necropsy findings of the dams at scheduled sacrifice on gd 21. There were no statistically significant differences in terminal body weight, gravid uterine weight or liver weight. A statistically significant decrease in corrected body weight was observed in the 1500 ppm group. Statistically significant decreases in corrected body weight gain were observed in the 1500 and 1000 ppm groups. In addition, although not statistically significant, corrected body weight gain in the 500 ppm group was slightly decreased. The statistically significant decrease in mean kidney weight of dams in the 1500 ppm group was not likely to be exposure-related. Rather, the decreased weight was related to the slightly decreased corrected body weight.

There were no effects of exposure on the number of ovarian corpora lutea, or on the number of total, viable, or nonviable implantations (early and late resorptions and dead fetuses) per litter. Percent preimplantation loss and sex ratio were equivalent across groups.

Dose descriptor:

NOAEC

Effect level:

5.4 mg/L air (analytical)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOEC

Effect level:

2.7 mg/L air (analytical)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: Decrease mean female fetal body weight, as well as increased incidences of external, visceral and skeletal variations were observed mainly in the 1000 and 1500 ppm dose levels.

Details on embryotoxic / teratogenic effects:
There were no statistically significant differences in individual external, visceral or skeletal malformations by category or of total malformations among all groups. There were no exposure-related increases in the incidence of variations by category (external, visceral or skeletal) or of total variations.

A statistically significant decrease in mean female fetal body weight but not in the body weight of combined sexes or in male fetal body weight was observed in the 1500 ppm group. In the 1000 ppm group, a statistically significant decrease in mean female fetal body weight was observed. The female fetal body weight data, however, were confounded by the fact that there was an increased litter size in dams exposed to 1500 ppm primary amyl acetate vapor and slight change in sex ratio between the control and 1000 ppm groups.

The incidences of one external fetal variation, ecchymosis in the head region, and one visceral variation, fetal atelectasis, were statistically significantly increased in the 1500 ppm group. The incidence of fetal atelectasis appeared to be increased in the 1000 ppm group as well. A total of 70 fetal skeletal variations were observed in this study. Statistically significant increases in the incidences of three of these were observed in fetuses in the 1500 ppm group; these included anterior arch of the atlas poorly ossified, thoracic centrum #9 bilobed, and majority of proximal phalanges of the hindlimb unossified. At 1000 ppm, a statistically significant increase in the incidence of majority of proximal phalanges of the hindlimb unossified was also noted. In addition, although not statistically significant, poorly ossified anterior arch of the atlas and thoracic centrum #9 bilobed appear to be increased in the 1000 ppm group. Poorly ossified anterior arch of the atlas and majority of proximal phalanges of the hindlimb unossified appear to be slightly increased in the 500 ppm group as well. Based on experience with the highly variable nature of these findings, the slightly increased incidences of skeletal variations alone observed in the low dose group were not considered to be of biological consequence. Statistically significant increases in other ossification sites were noted for the 1000 and 500 ppm exposure groups but these were not considered to be exposure-related due to the lack of a concentration-response relationship.

Dose descriptor:

NOAEC

Effect level:

>= 8.1 mg/L air (analytical)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

no effects observed

Developmental effects observed:

no

Maternal and Litter Values in Rats Exposed to Primary Amyl Acetate
	Concentration of Primary Amyl Acetate
	0 ppm	500 ppm	1000 ppm	1500 ppm
Number females on study	25	25	25	25
Number females pregnant	24	24	24	24
Maternal food consumption (gm)
Pretreatment (GD 0-6)	12.94±0.89	13.20 ± 1.17	13.62 ± 1.41	12.98 ± 0.69
Treatment (GD 6-15)	13.60 ± 0.77	13.59 ± 0.57	13.36 ± 0.96	13.28 ± 0.61
Post-treatment (GD 15-21)	16.69 ± 0.92	15.87 ± 1.15**	15.97 ± 0.82**	15.50 ± 0.84**
Final corrected body weight^1	180.84 ± 5.34	177.59 ± 6.74	177.38 ± 5.95	175.33 ± 5.13**
Body weight gain (corrected)^2	26.05 ± 5.34	22.93 ± 6.83	22.66 ± 5.33*	20.74 ± 4.69**
Number pups per litter	7.8 ± 3.28	8.6 ± 2.65	7.9 ± 3.22	9.1 ± 2.74
Total number of pups	187	207	190	219
Fetal body weights per litter (gm)
Males	4.533 ± 0.264	4.472 ± 0.187	4.458 ± 0.337	4.367 ± 0.208
Females	4.240 ± 0.237	4.227 ± 0.208	4.091 ± 0.175*	4.061 ± 0.151*
1: Corrected body weight = body weight at sacrifice minus gravid uterine weight 2: Body weight change (corrected) = corrected body weight – initial body weight * Significantly different from control group, p< 0.05. ** Significantly different from control group, p <0.01

 

Summary of Variations Observed in Rat Fetuses^1
	Concentration of Primary Amyl Acetate
	0 ppm	500 ppm	1000 ppm	1500 ppm
Total variations
Number affected pups	149 (79.7%)	158 (76.3%)	159 (83.7%)	175 (79.9%)
Number affected litters	24	24	24	24
External variations^2
Number affected pups	83 (44.4%)	75 (36.2%)	91 (47.9%)	105 (47.9%)
Number affected litters	23	23	22	22
Soft tissue variations^3
Number affected pups	24 (24.0%)	30 (27.5%)	42 (41.2%)	37 (32.2%)
Number affected litters	14	13	20	18
Skeletal variations^4
Number affected pups	87 (100%)	98 (100%)	88 (100%)	104 (100%)
Number affected litters	23	24	23	23
1: Findings are for the number of affected fetuses, followed by percent affected relative to total number of fetuses examined. No findings were significant as compared to controls (p < 0.05) 2: All fetuses were examined for external variations 3: Approximately 50% of each litter were examined for viscerally for soft tissue defects 4: Approximately 50% of each litter were examined for skeletal defects

 

Percent Incidence of Certain Variations
Skeletal Variation	Concentration of Primary Amyl Acetate				Historical Control Range^1
	0 ppm	500 ppm	1000 ppm	1500 ppm
Anterior Arch of the Atlas
Poorly ossified	39.1 (F)2	51	53.4	40.4	39-86
	73.9 (L)2	83.3	82.6	100.0*	74-100
Unossified	2.3	1	8	6.7	2-4
	8.7	4.2	30.4	30.4	9-17
	3.4	3.1	3.4	1	1-6
	13	12.5	13	4.3	4.5-25
Bilobed	6.9	3.1	4.5	9.6	0-7
	21.7	12.5	13	34.8	0-22
Hind Limb Phalanges
Some poorly ossified^3	67.8 (F)^2	72.4	67	67.3	30-68
	100 (L)^2	95.8	91.3	100	59-100
Majority poorly ossified^3	4.6	5.1	1.1	4.8	5-11
	17.4	20.8	4.3	21.7	17-27
Some unossified	75.9	76.5	59.1	62.5	32-76
	82.6	100*	91.3	95.7	53-96
Majority unossified	5.7	14.3	21.6	20.2	6-28
	17.4	41.7	56.5*	60.9*	17-61
All unossified	5.7	3.1	14.8	15.4	6-31
	21.7	8.3	43.5	47.8	22-50
Thoracic Centra 9-12
#9	0 (F)^2	0	0	1
	0 (L)^2	0	0	4.3
#9 Bilobed	1.1	5.1	6.8	6.7	1-17
	4.3	16.7	26.1	30.4*	4-58
#10	0	0	0	1
	0	0	0	4.3
#10 Bilobed	10.3	24.5	20.5	14.4	6-47
	34.8	75.0*	56.5	56.5	24-92
#11	0	1	0	1	0-1
	0	4.2	0	4.3	0-5
#11 Bilobed	28.7	38.8	29.5	33.7	4-64
	69.6	79.2	60.9	82.6	12-100
#12	0	1	0	1
	0	4.2	0	4.3
#12 Bilobed	19.5	21.4	17	18.3	4-47
	56.5	58.3	56.5	47.8	6-96
1: Historical control data from five studies performed by the same laboratory 2: Top number = % affected fetuses, bottom number = % affected litters 3: Majority = 5 or more phalanges affected; some = 1 to 4 phalanges affected *Statistically different from concurrent controls (p < 0.05)

 

Percent incidence of visceral and external variations
Variation	Concentration of Primary Amyl Acetate
	0 ppm	500 ppm	1000 ppm	1500 ppm
Fetal atelectasis	10.0^1	10	21.5	17.4
	29.2^1	25	50	62.5*
Ecchymosis-head	1.6^1	2.4	21.6	17.4
	12.5^1	16.6	29.2	41.7*
1: top number = % affected fetuses, bottom number = % affected litters *statistically different from concurrent controls, p < 0.05

 

Executive summary:

Four groups, each consisting of 25 timed-pregnant Fischer 344 rats were exposed to primary amyl acetate vapor for 6 hours/day on gestational days (gd) 6 through 15. Target concentrations of primary amyl acetate were 0 (control), 500, 1000, and 1500 ppm. Clinical observations were conducted daily, and body weights were measured on gd 0, 6, 9, 12, 15, 18 and 21. Maternal food consumption was measured at 3 day intervals throughout gestation, gd 0-21. At scheduled sacrifice on gd 21, the dams were evaluated for liver, kidney, and gravid uterine weights. Ovarian corpora lutea of pregnancy were counted and the status of implantation sites (i.e., resorptions, dead and live fetuses) were identified and recorded. Live fetuses were dissected from the uterus, counted, weighed, sexed, and examined for external abnormalities. Approximately one-half of the live fetuses in each litter were examined for visceral malformations and variations. These fetuses were then decapitated and the heads fixed in Bouin's solution; serial free-hand sections of the heads were examined for soft tissue craniofacial malformations and variations. The remaining half of the fetuses (intact) in each litter were eviscerated, fixed in alcohol, stained with alizarin red S and examined for skeletal malformations and variations.

Maternal toxicity was observed in the 1500 ppm group and was evidenced by decreases in gestational body weight gains, corrected body weight, and corrected body weight gain. Food consumptions were decreased in all primary amyl acetate-exposed groups during the postexposure period. The biological significance of the decreased food consumption is not clear due to the lack of the same finding during the exposure period. Corrected body weight gains were slightly decreased in the 500 and 1000 ppm groups. However, the biological significance of these decreases is not clear due to the lack of any other changes in body weight or body weight gains during the study and the small magnitude of the changes. In addition, the decreased corrected body weight gain observed in the 500 ppm group was confounded by the slightly increased number of viable implants.

However, these highly variable findings observed in the 500 ppm group were not observed in conjunction with reduced fetal body weights and were not considered to be of biological consequence.

Exposure of pregnant rats to primary amyl acetate vapor during organogenesis resulted in maternal toxicity and reduced or delayed development at concentrations of 1000 and 1500 ppm. Maternal toxicity as indicated by decreased corrected body weight gain, was also observed even at the lowest concentration; however, the biological relevance of this decrease is unclear. Thus, the "no-observed-adverse-effect level" (NOAEL) of 1000 ppm was noted for maternal toxicity. The no-observed-effect level was 500 ppm for developmental toxicity based on increased variations. Since no malformations were observed, the NOAEC was 1500ppm, the highest dose tested.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

8 100 mg/m³

Species:

other: rat and rabbit

Additional information

Developmental toxicity of the reaction mass of 2 -methylbutyl acetate and pentyl acetate was examined in study following the OECD guideline 414 (UCC, 1994). 25 Fischer 344 rats per dose were exposed to 500, 1000 and 1500 ppm (corresponding to 2.7, 5.4 and 8.1 mg/l air, respectively) for 6 hours/day on days 6 - 16 of gestation. On gestation day 21, the dams were sacrificed, and ovaries and uterine content as well as fetuses were examined. Maternal toxicity was observed in the 1500 ppm group as evidenced by decreases in gestational body weight gain, corrected body weight, and corrected body weight gain. Corrected body weight gains for females in the 1000 and 1500 ppm groups were reduced by 13.3% and 20.4%, respectively, relative to controls. Maternal food consumption was significantly reduced in all treated groups during the post-treatment interval, thus the NOAEC (maternal toxicity) was determined as ca. 5.4 mg/l (Union Carbide Corporation, 1994).

A statistically significant decrease in mean female fetal body weight but not in the body weight of combined sexes or in male fetal body weight was observed in the 1500 ppm group. In the 1000 ppm group, a statistically significant decrease in mean female fetal body weight was observed. The female fetal body weight data, however, were confounded by the fact that there was an increased litter size in dams exposed to 1500 ppm primary amyl acetate vapor and slight change in sex ratio between the control and 1000 ppm groups. Accompanying the reduced female fetal body weights were increases in one minor fetal skeletal variation at 1000 ppm, and three minor skeletal variations at 1500 ppm. These specific variations were poorly ossified anterior arch of the atlas (1500 ppm), unossified majority of hindlimb phalanges (1000 and 1500 ppm), and bilobed thoracid centrum #9 (1500 ppm). There were two variations that displayed a dose response when evaluated on a percent litter affected basis. However, the data were statistically significant only at the highest dose. These were the external variation, ecchymosis of the head, and the visceral variation, fetal atelectasis. Due to the described effects the NOEC was therefore 2.7 mg/l. No exposure-related malformations were observed at any dose level; therefore the NOAEC was >/= 8.1 mg/l.

Additionally, Union Carbide conducted a developmental toxicity/teratogenicity study with New Zealand White rabbits (1994). 15 rabbits per dose were exposed to 500, 1000 and 1500 ppm (corresponding to ca. 2.7, 5.4 and 8.1 mg/l air, respectively) for 6 hours/day on days 6 - 18 of gestation. On gestation day 29, the dams were sacrificed and ovaries and uterine content as well as fetuses were examined. Maternal body weight losses during the first week of exposure, reduced maternal food consumption, and reduced maternal body weight gain in females exposed to 1500 ppm were observed. Fetal examination produced no evidence of developmental toxicity or fetotoxicity in any of the exposure groups. Therefore, the NOAEC for maternal toxicity is ca. 5.4 mg/l; the NOAEC for developmental toxicity and teratogenicity is >/= 8.1 mg/l.

Justification for classification or non-classification

Exposure to the reactions mass of 2 -methylbutyl acetate and pentyl acetate did not cause malformations in rats and rabbits. Furthermore, the registered substance did not impair fertility in rats.

Consequently no classification is required for reproductive toxicity according to GHS.