Reaction mass of 2-methylbutyl acetate and pentyl acetate

Endpoint summary

Description of key information

90 -day, inhalation, rat: NOAEC = 500 ppm, no effects up to highest dose (sim. to OECD 413, GLP, UCC, 1985)

short term study, gavage, rat: NOAEL: 1000 mg/kg bw no effects up to highest dose (OECD 422, GLP, BASF 2020)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same 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

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)

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

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

Observations and examinations performed and frequency:

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.

Sacrifice and pathology:

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.

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.

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

Description (incidence and severity):

Absolute organ weights
When compared to control group 0 (=100%), the mean absolute weights of the uterus were significantly increased in test groups 2 and 3:

Test group 1 2 3
(mg/kg bw/day) (100) (300) (1000)
Uterus -9.1% +7.3%* +22.6%*
*p <= 0.05; **p <= 0.01

All other mean absolute weight parameters in females and all weight parameters in males did not show significant differences when compared to the control group 0.

Relative organ weights
When compared to control group 0 (=100%), the mean relative weight of the uterus was significantly increased in test group 3:
Test group 1 2 3
(mg/kg bw/day) (100) (300) (1000)
Uterus -9.8% +8.2% +19.8%*
*p <= 0.05; **p <= 0.01
All other mean relative weight parameters in females and all weight parameters in males did not show significant differences when compared to the control group 0.

The increased mean absolute and relative uterus weights in test group 3 and the increased mean absolute uterus weight in test group 2 were regarded to be incidental as they were within historical controls and there was no histopathological correlate. Historical controls ranged from 0.527 – 1.699 g for absolute weights and for relative weights from 0.211 % to 0.666 %. In this study, test group 2 showed a mean absolute weight of 0.66 g and test group 3 a mean absolute/relative weight of 0.76 g/0.32 % which are well within the historical control range.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Most findings occurred individually apart from 2 foci in the glandular stomach in test group 2 females. As no foci in the glandular stomach occurred in any other group all recorded findings were considered to be incidental or spontaneous in origin and without any relation to treatment.

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.

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

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.

Endpoint conclusion

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

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 May - 7 Sept 1984

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day 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

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Species, Source, and Quality Control
One-hundred four (104) male and one-hundred five (105) female rats [COBS CDF@ (F-344/Cr1BR)], 34 days of age, were received on April 16, 1984 from Charles River Breeding Laboratories, Inc. (Kingston, NY). Shortly after delivery, random fecal samples were collected and examined for intestinal parasites by zinc sulfate flotation. Results of the fecal tests were negative. Blood samples were collected from three male and three female rats for serology prior to sacrifice. A gross examination was performed on the same rats used for serology evaluations and the liver, heart, kidneys, salivary glands, nasal turbinates, lungs, trachea, spleen and cervical lymph nodes were fixed and examined microscopically. Physical examination, parasitology,
serology, and histology on the quality control animals were within expected limits for this strain and species. Body weight determinations and clinical
and ophthalmologic examinations of all animals were performed prior to the initiation of exposures.

Animal Husbandry
The rats were housed two per cage in stainless steel, wire-mesh cages (23.5 cm x 40 cm x 18 cm high). All animals were housed separately by test group and sex. A layer of Deotized Animal Cage Board* (Shepherd Specialty Papers, Inc., Kalamazoo, MI) was placed under each cage. The room temperature and relative humidity were recorded continuously (Hygrothermograph Seven-Day Continuous Recorder, Model #8368-00, Cole-Parmer Instrument Company, Chicago, IL). The animals were kept on a 12-hour photoperiod throughout the study.

During non-exposure periods, water (Municipal Authority of Westmoreland County, Greensburg, PA) supplied by an automatic watering system, and powdered food (Purina Certified Rodent Chow #5002, Ralston Purina Company, St. Louis, MO) were available ad libitum. Analyses of the food and water showed no contaminants at concentrations high enough to interfere with the outcome of the study.

During the exposures, the animals were housed two per cage, separated by sex and test group, in stainless steel wire-mesh cages, 12.5 cm x 20 cm x 17 cm high. Food and water were withheld during exposure.

Group Assignment
The body weight and physical condition of all animals were monitored for approximately two weeks prior to placement into exposure groups. Animals were assigned to three test groups and an air-exposed control group (20 per sex per group) using a computer-based (Wil Laboratories Toxicology Data Management System) randomization program. At the time of group assignment, only animals with body weights within two standard deviations of the group mean for each sex were used in the study. Any animal in poor health was rejected from group assignment.

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: Not applicable.

Details on inhalation exposure:

Inhalation Chamber Description and Operation
The inhalation chambers used in the study were constructed from stainless steel with glass windows for animal observation. The volume was approximately 1330 liters and the airflow was approximately 300 L/min (13-14 air changes per hour). Chamber temperature and relative humidity were recorded using an Ashcroft dial-type thermometer and an Abbeon Certified Hygrometer Model AB167B (Abbeon Cal, Inc., Santa Barbara, CA), respectively. Temperature and relative humidity measurements were recorded at least four times per exposure.

Target Concentrations and Exposure Regimen
The animals were acclimated to the exposure chambers (air-only exposure) for two days prior to the initiation of the PAA exposure regimen. Target concentrations of 500, 300, 100, and 0 (control) ppm primary amyl acetate were selected for this study by the Sponsor after results of a preliminary 9-day inhalation study had been reviewed. The rats (55 days of age at initiation of exposures) were exposed for six hours per day, five days a week for thirteen weeks, except for the fourth week when there were 4 exposure days (holiday). During the fourteenth week there were 3 days of exposure for males sacrificed the following day and four days of exposure for all females and male recovery rats. Control (air-exposed) animals were handled in an identical manner as the PAA-exposed animals.

The position of cages was rotated weekly in a predetermined pattern within each chamber to compensate for any possible, but undetected, variations in chamber conditions.

Vapor Generation
The primary amyl acetate liquid 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 -a l . (1975). Temperatures in the evaporator were maintained near the minimum temperature required to vaporize the liquid. The resultant vapor was carried into the chamber by a Countercurrent air stream that entered the bottom of the evaporator.

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

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chamber concentrations of primary amyl acetate were analyzed approximately once each hour by gas chromatography.

Duration of treatment / exposure:

6 hrs/day

Frequency of treatment:

5 days/week except for holidays

Remarks:

Doses / Concentrations:
0, 100, 300 and 500 ppm
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
0, 101±3.8, 303±8.8, and 509±16.4 ppm (corresponding to approx. 0.54, 1.61 and 2.71 mg/l, calculated assuming test substance molecular weight of 130.19 g/mol)
Basis:
analytical conc.

No. of animals per sex per dose:

20 rat/sex/dose level

Control animals:

yes, concurrent no treatment

Details on study design:

Four groups, each consisting of twenty (20) Fischer 344 rats per sex were exposed for 6 hours per day, 5 days per week for 14 weeks to vapors of primary amyl acetate (PAA) at target concentrations of 0, 100, 300 or 500 ppm. Actual mean concentrations obtained for this study were 101, 303, and 509 ppm PAA. The following parameters served as monitors for toxic effects: clinical observations, body weight, food and water consumption, ophthalmology, hematology, clinical chemistry, urinalysis, organ weights, and macroscopic and microscopic tissue evaluations.

Positive control:

No data.

Observations and examinations performed and frequency:

Animal Observation
All animals were observed prior to, during, and following each exposure for signs of toxic effects. Animals were observed once a day on weekends and holidays during the fourteen week exposure regimen. The recovery animals were observed once a day.

Body Weights
All animals were weighed just prior to the initiation of the first exposure. These values were used as the pre-exposure reference weights (labeled study day "zero") and were substracted from each subsequent weight to determine the change in body weight. The animals were weighed weekly during
the exposure period and preceding sacrifice. Body weights for animals held during the epostexposure period were determined weekly and just prior to sacrifice.

Food and Water Consumption
Ten rats per sex per exposure group were individually housed in round polycarbonate metabolism cages with stainless steel, wire-mesh bottoms,
approximately 20 cm diameter x 11 cm high (Nalge Company, Rochester, NY). Food and water consumption were measured for approximately 15 1/2 or 16 hours for male and female rats, respectively, following sixty-seven exposures for the male rats and sixty-eight exposures for the female rats. Food and water consumption evaluations were not conducted on the recovery animals.

Ophthalmologic Evaluation
Prior to the first exposure and at sacrifice, the anterior chambers of the eyes of each animal were examined by a veterinarian using an external light
source and a magnifying lens. At the recovery sacrifice, the rats were examined by a veterinarian using a direct light ophthalmoscope to observe the
anterior chamber of the eye.

Sacrifice and pathology:

Blood Analysis
Serum chemistry and hematologic evaluations were performed on blood samples collected from eighty rats (10 per sex per exposure group) just prior to sacrifice on the day following the last exposure and from eighty recovery rats (10 per sex per group) just prior to sacrifice. Blood was obtained from the orbital sinuses of methoxyflurane- anesthetized animals. Water was available to all animals ad libitum during blood collection and necropsy.

Urinalysis
Urine was collected while the rats were in the metabolism cages. Food and water were available ad libitum.

Necropsy and Pathology
Ten male rats per exposure group were sacrificed on August 9; ten female rats per exposure group were sacrificed on August 10, 1984. The recovery rats, ten males and ten females per exposure group were sacrificed on September 6 and 7, 1984, respectively. The rats were killed by exsanguination via the brachial blood vessels following anesthesia with methoxyflurane. Gross examinations were performed and selected tissues were saved in neutral buffered 10% formalin for possible future histologic evaluation. Histologic evaluation was performed on selected tissues from animals in both the 500 ppm and control groups.

Tissues collected at necropsy included:
adipose tissues
adrenals*
aorta
bone and bone marrow - femoral, sternal*, vertebral
bone marrow smear
brain* - brain stem*, cerebellum*, cerebrum*
cervix
ears
epididymides*
esophagus
eyes
fallopian tubes
heart*
intestine - large (3 levels) small (3 levels)
kidneys*
lacrimal glands
larynx*
liver*
lungs
lymph nodes - cervical*, mesenteric, thoracic (mediastinal)
mammary tissue
muscle - gastrocnemius*
nasal turbinates*
nerve - plantar, sciatic*, tibial
ovaries
pancreas
parathyroids*
pituitary*
prostate (and associated sex glands)
salivary glands
skin (flank)
spinal cord (lumbosacral section)
spleen*
stomach
testes*
thymus*
thyroids*
trachea*
urinary bladder*
uterus
vagina
Zymbal ' s gland
any lesions
* tissues examined histopathologically

Organ Weights
The brain, liver, kidneys, spleen, lungs, and testes (males only) from all animals were weighed at sacrifice. Organ weights were recorded as absolute
weights and as relative (as a percentage of body weight) weights.

Other examinations:

No additional information available.

Statistics:

Results of quantitative continuous variables were intercompared among the three exposure groups and one control group by use of Bartlett's homogeneity of variance (Sokal and Rohlf, 1969), analysis of variance (ANOVA) (Sokal and Rohlf, 19691, and Duncan's multiple range tests (Snedecor and Cochran, 1967). The latter was used to delineate which exposure groups differed from the control, when F from the analysis of variance was significant. If Bartlett's test indicated heterogeneous variances, all groups were compared by an ANOVA for unequal variances (Welch or Brown and Forsythe, 1974) followed if necessary by t-tests. Corrected Bonferroni probabilities were used for t-test comparisons. Statistical procedures for hematologic continuous variables were similar. The fiducial limit of 0.05 (two-tailed) was used as the critical level of significance for all comparisons.

References
Sokal, R. R. and Rholf, F.J. (1969). Biometry, W. H. Freeman and Company, San Francisco, pp. 299-340 and 369-371.

Snedecor, G. W. and Cochran, W. G. (1967). Statistical Methods, Iowa State University Press, Ames, 10, 274-275.

Brown, M. B. and Forsythe, A. B. (1974). The Small Sample Behavior of Some Statistics Which Test the Equality of Several Means. Technometrics 16,
129-132.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Control group appeared to have faster growth rate than other control animals of same age which resulted in an apparent effect in rats exposed to PAA.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Details on results:

Chamber Concentration, Temperature and Relative Humidity
The target concentrations were 100, 300 and 500 ppm of PAA vapor. Control animals were exposed to filtered air only. Gas chromatographic analyses of the chamber atmospheres resulted in mean (+/- standard deviation) concentrations of 101 (+/- 3.8), 303 (+/- 8.8) and 509 (+/- 16.4) ppm, respectively. The mean analytical to nominal concentration ratios were 0.97, 0.99 and 0.98, respectively. No amyl acetate was detected in the control chamber.

During the exposures, the daily mean chamber temperature and relative humidity ranged between 73-79F and 42-58%, respectively.

Housing condition
The temperature and relative humidity in the animal housing room ranged between 68-79F and 35-70%, respectively, throughout the study period.

Animal Observations
There were no exposure-related clinical signs observed for either sex of any of the exposure groups.

On June 27, 1984, Exposure Day 38, there was an error made in placement of the 100 and 300 ppm group males in the chambers. The 100 ppm group males were placed in the 300 ppm exposure chamber; the 300 ppm group males were placed in the 100 ppm exposure chamber. The mean exposure concentrations were 99.4 and 306 ppm in the 100 and 300 ppm exposure chamhers, respectively. The error occurred only on Exposure Day 38. On June 29, 1984, all animals were weighed and individually observed for clinical signs. There was neither a perturbation in body weight gain for the 100 ppm exposure group males nor were any clinical signs present in those rats. Thus, this error did not alter the interpretation of the results of this study.

Mortality
There were no unscheduled deaths during the study.

Body Weights
All PAA exposure groups of males had body weight gains that were statistically significantly lower than the controls by the second week of the exposure, with the 300 and 500 ppm groups also exhibiting significantly lower weight gains during the first week of exposures. The lower weight gains persisted throughout the entire exposure period for all three PAA exposure groups, and the absolute body weights for all PAA-exposed groups of males were also significantly lower than controls at the end of the exposure regimen. A significant decrease in body weight gain was observed during the first week of the recovery period (days 95-102) for the 100 and 300 ppm group males. Although the body weight gains were still below those of controls for the last three weeks of the recovery period, the difference was not statistically significant. There were also no statistically significant differences for the absolute body weights between groups of males at the end of the recovery period. All PAA-exposed groups of males had similar body weight gains throughout the study, i. e., there were no concentration-response decreases in body weight gain.

For females, there were no exposure-related effects observed on body weight gain or absolute body weight during the study. The 100 ppm females had significantly higher body weight gains and/or absolute body weights at various intervals during the exposure period, but not during the recovery period.

Food and Water Consumption
There were no significant differences in food or water consumption observed in either sex of the study groups.

Ophthalmologic Evaluations
There were no exposure-related ocular effects observed during the study.

Blood Analysis and Urinalysis
There were no statistically significant differences between control and PAA-exposed groups of animals for any of the clinical chemistry,
hematology or urinalysis parameters evaluated for the study.

Necropsy
There were no exposure-related macroscopic lesions observed during the exposure or recovery periods.

Organ Weights
There were signficantly decreased absolute liver and testes weights in all groups of PAA-exposed males at the end of 14 weeks of exposure. Significantly increased absolute brain and spleen weights were observed in all groups of PAA-exposed females, compared to controls, at the end of the 14-week exposure period. There were significantly increased relative brain weights in the 100 and 500 ppm exposure group males at the end of the exposure period. There were no statistically significant differences observed for any organ weights for the study groups at the end of the recovery period.

Anatomic Pathology
There were no exposure-related differences between the control and 500 ppm exposure groups.

Dose descriptor:

NOAEC

Effect level:

2.66 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment related effects observed at any dose level during this study (original value: 500 ppm)

Critical effects observed:

no

Conclusions:

The exposure of rats to primary amyl acetate at air concentrations up to 509 ppm did not result in adverse effects on weight gain or food and water consumption. Toxicologically significant changes were not noted in clinical examination of blood or urine. Clinical signs of toxicity were not observed. Finally, there was no evidence of organ or tissue injury in animals exposed for 90 days to air concentrations of primary amyl acetate up to 509 ppm.

Executive summary:

Four groups, each consisting of twenty (20) Fischer 344 rats per sex were exposed for 6 hours per day, 5 days per week for 14 weeks to vapors of primary amyl acetate (PAA) at target concentrations of 0, 100, 300 or 500 ppm. Actual mean concentrations obtained for this study were 101, 303, and 509 ppm PAA. The following parameters served as monitors for toxic effects: clinical observations, body weight, food and water consumption, ophthalmology, hematology, clinical chemistry, urinalysis, organ weights, and macroscopic and microscopic tissue evaluations. There were no mortalities during the study and no exposure-related effects were observed in any of the parameters evaluated for this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

2 660 mg/m³

Study duration:

subchronic

Species:

rat

Additional information

Inhalation exposure

In a subchronic inhalation toxicity study performed equivalent to OECD 413 (UCC, 1985 / TSCAT OTS0557477) four groups of twenty Fischer 344 rats per sex were exposed to target vapor concentraions of 100, 300, or 500 ppm of the registered substance for 6 hours per day, 5 days per week for 14 weeks. Actual obtained concentrations were 101, 303, and 509 ppm. The following parameters served as monitors for toxic effects: clinical observations, body weight, food and water consumption, phthalmology, hematolog, clinical chemistry, urinalysis, organ weights, and macroscopic and microscopic tissue evaluations. there were no mortalities during the study and no exposure-related effects were observed in any of the parameters evaluated for this study. Consequently, the NOAEC was 500 ppm for males and females.

Additionally, neurotoxicity was assessed by Gill et al. (2000, see IUCLID section 7.9.1 for details). The study followed USEPA (1985) Test Guidelines for neurotoxicity. Rats were exposed for 6 hours per day, 5 days per week and 14 weeks, to vapours of test substance at target concentrations of 0, 300, 600 or 1200 ppm. Treatment groups consisted o 15 rats per sex for control and high dose and 10 rats per sex for low and mid dose groups. Evaluations included clinical observations before, during and after each exposure, weekly detailed clinical examinations, weekly body weight and food consumption measurements and monthly functional observational battery and motor activity measurements conducted on non-exposure days. Neuropathology examination of the central and peripheral nervous system was performed at the end of the study. Exposure did not result in overt clinical signs of toxicity or changes in body weight or food consumption. Transient, subtle decreases in general activity level during the 6 -h exposure period were noted for animals in the 600 and 1200 ppm groups during the first 2 weeks of the study. Functional observational battery evaluations, automated motor activity measurements and neuroanatomy were unaffected by exposure. The no observed-effect concentration for subchronic neurotoxicity was at least 1200 ppm.

 

Oral exposure

In an OECD guideline 422 compliant study (BASF 2020), 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.

Justification for classification or non-classification

Based on the above results after repeated exposure for up to 90 days, there is no need for classification according to the current regulations.

GHS classification (REGULATION (EC) No 1272/2008 (CLP)):no classification required.