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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Principles of method if other than guideline:
The study was conducted according to OECD 421 guideline with some additional parameters of an OECD 422 guideline study.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Benzaldehyde, dodecylhydroxy-,oxime, branched (solvent-free and dodecylphenol-depleted)
- Physical state: Liquid, highly viscous, golden brown
- Analytical purity: 99.2 corrected area-%
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: (P)10-12 wks (11-13 wks beginning of treatment)
- Weight at study initiation: (P) Males: 354.1 g - 382.8 g; Females: 191.6 g - 212.4 gdy:
- Housing: During the study period, the rats were housed individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area of about 800 cm²), with the following exceptions:
- During overnight matings, male and female mating partners were housed together in Makrolon type M III cages.
- Pregnant animals and their litters were housed together until PND 4.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: about 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance solutions in corn oil were prepared at the beginning of the administration period and thereafter in intervals, which took into account the analytical results of the stability verification.

For the preparation of the administration solutions the test substance was weighed in a calibrated beaker depending on the dose group, topped up with corn oil and subsequently intensely mixed with a magnetic stirrer until it was completely dissolved.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the test substance was completely miscible with corn oil
- Concentration in vehicle: 0, 0.75, 2.50 and 7.50 g/100 ml
- Amount of vehicle (if gavage): 4 ml/kg bw/day
Details on mating procedure:
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 07.00 - 09.00 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 "GD 0" and the following 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 corn oil for a period of 7 days at room temperature were carried out prior to the start of the study.

Given that test substance was completely miscible with corn oil, solutions were considered to be homogenous without further analysis.

Samples of the test substance solutions were sent to the analytical laboratory once at the beginning of the study for verification of the concentrations.

Of each sample, one additional reserve sample was retained. Details of the sampling schedule were recorded with the raw data.
Duration of treatment / exposure:
50 days (males) and 56 days (females)
Frequency of treatment:
once daildy
Details on study schedule:
N/A
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 30, 100 and 300 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on a range-finder
- Rationale for animal assignment (if not random): random
Positive control:
N/A

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
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.

A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. 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.

BODY WEIGHT: Yes
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.

The following exceptions are notable for the female animals:
- 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 4.

Females without positive evidence of sperm, without litter or waiting for necropsy, were weighed weekly. These body weight data were solely used for the calculations of the dose volume.

FOOD CONSUMPTION: Yes
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) and during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on gestation days (GD) 0 - 7, 7 - 14, and 14 - 20.
- Food consumption of F0 females, which gave birth to a litter was determined on PND 1 - 4.

Food consumption was not determined in females without positive evidence of sperm during gestation periods and in females without litter during lactation and post-mating period.
Oestrous cyclicity (parental animals):
N/A
Sperm parameters (parental animals):
Immediately after necropsy and organ weight determination the right testis and cauda epididymis were taken from all male animals.

Sperm motility examinations were carried out in a randomized sequence. Sperm head count (testis and cauda epididymis) were evaluated in all animals.

Parameters examined:
- Sperm motility
- Sperm morphology
- Sperm head count (cauda epididymis)
- Sperm head count (testis)
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 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 section “Necropsy observations”.

The number and percentage of dead pups on the day of birth (PND 0) and of pups dying between PND 1-4 (lactation period) were determined. Pups which died accidentally or were 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 day 4.

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. The sex of the pups was finally confirmed at necropsy.

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.

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).

“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.


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 was given to the reproductive organs.

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

1. Anesthetized animals
2. Adrenal glands
3. Epididymides
4. Liver
5. Kidneys
6. Ovaries
7. Pituitary gland
8. Prostate
9. Seminal vesicle (including coagulation glands)
10. Spleen
11. Testes

Organ/Tissue fixation of parental animals
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
4. Coagulating glands
5. Epididymis, left (modified Davidson’s solution)
6. Liver
7. Kidneys
8. Ovaries (modified Davidson’s solution)
9. Oviducts
10. Pituitary gland
11. Prostate gland
12. Seminal vesicles
13. Spleen
14. Testis, left (modified Davidson’s solution)
15. Vagina
16. Uterus
The ovaries of the animal that died were fixed in 4% neutral-buffered formaldehyde solution.

3.10.4. Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings was performed in all dose groups on the following organs:

1. Testis, left
2. Epididymis, left
3. Ovaries

The animal that died was processed histotechnically and assessed like control animals.

The organs were trimmed according to the “Revised guides for organ sampling and trimming in rats and mice”.

A correlation between gross lesions and histopathological findings was attempted.
Postmortem examinations (offspring):
All pups with scheduled sacrifice on PND 4 were sacrificed under isoflurane anesthesia with CO2. All pups were examined externally and eviscerated; their organs were assessed macroscopically.

All stillborn pups and all pups that died before PND 4 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.
Statistics:
Detailed statistical analyses were conducted
Reproductive indices:
The following indices were calculated: Male mating index, Male fertility index, Female mating index, Female fertility index, Gestation index,
Offspring viability indices:
Live birth index, Postimplantation loss, Pup viability index, Pup sex ratio

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One F0 female of test group 2 (No. 123 - 100 mg/kg bw/d) was sacrificed moribund because of piloerection, pale skin, blood in bedding and inability to deliver.
No other mortalities were noted in any of the groups.

With one exception (No. 123 - 100 mg/kg bw/d), no clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any male or female F0 generation parental animals during the whole study period.

Several male and female animals of dose groups 3 and 2 (300 and 100 mg/kg bw/d) showed salivation after treatment during the whole study period. This transient salivation for a few minutes immediately after treatment was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity.

All sperm positive and negative high-dose females (Nos. 131 - 140) and one sperm negative low-dose female (No. 116) did not become pregnant.

One mid-dose female (No. 128) had a complete litter loss on PND 1

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
The high-dose parental males had statistically significantly lower body weights on premating days 7 - 13, during the whole mating and post-mating period (up to 7%, about 7% and up to 13% below the concurrent control values, respectively). Their body weight change was statistically significantly lower than the concurrent control values (over 100% respectively) during the whole premating and post-mating period (0.7 g vs. 30.4 g in control and -9.4 g vs. 11.6 g in control, respectively).

Mean body weights and mean body weight change of the mid- and low-dose parental males were comparable to the concurrent control group during the entire study period.

The mid-dose parental females had statistically significantly lower body weights on GD 14 and 20 (up to 14% below the concurrent control values) as well as statistically significantly lower body weight changes during GD 7 – 20 (up to 47% below concurrent controls) and GD 0 - 20 (about 38% below the concurrent control group).

The high-dose F0 females did not show any test substance-related changes in mean body weights or mean body weight changes during the premating phase, the only phase in which these parameters were measured.

Low-dose F0 females did not show any test substance-related changes in body weight parameters throughout the whole treatment period.

Food consumption of the high-dose F0 males (300 mg/kg bw/d) was statistically significantly below the concurrent control values during the whole premating period (up to 24%).

The mid- and low-dose F0 males (100 and 30 mg/kg bw/d) did not show any test substance-related changes in food consumption during the whole treatment period.

Food consumption of the mid-dose F0 females was statistically significantly below the concurrent control value during the lactation period (PND 1 – 4, about 40%), but not in any other study phase.

The high-dose F0 females did not show any test substance-related changes in food consumption during the premating phase; moreover, due to the lack of pregnancies in this dose group, this was the only phase in which food consumption was measured.

Low-dose F0 females did not show any test substance-related changes in food consumption throughout the whole treatment period.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
In males of test group 3 (300 mg/kg bw/d) more abnormal sperms were found in the cauda epididymidis (headless sperms, abnormal hook of the heads or bent heads, broken or missing tails and changes of single sperms at the head and the tail) and the sperm head counts were decreased in males of test groups 1, 2 and 3 (30, 100 and 300 mg/kg bw/d).

Concerning the motility of the sperms and the sperm head counts in the testis no treatment-related effects were observed.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Males:
For all F0 parental males except males No. 16, 32 and 36, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in control and test group 2, 90% in test group 1 and 80% in test group 3. The lower mating index in group 3 is still within the historical control range (80-100%). However, in the light of the decreased sperm quality and pathological findings in reproductive organs in this dose group, a relationship to the treatment is likely.

No high-dose male (300 mg/kg bw/d - Nos. 31 - 40) was able to produce implants. This infertility is associated with reduced sperm quality and pathological alterations in the sexual organs (for details see 4.3.3. Sperm parameters and 4.4. Pathology). These findings are considered to be related to the test item.

Fertility was proven for most of the F0 parental males in test groups 0-2 within the scheduled mating interval for F1 litter. One low-dose male (30 mg/kg bw/d - No. 16) did not generate implants, this is, however, considered to be unrelated to treatment. Thus, the male fertility index was 0% in test group 3, 100% in test group 2 and the control and 90% in test group 1.

Females:
The female mating index calculated after the mating period for F1 litter was 100% in test group 2 and the control, 90% in test group 1 and 80% in test group 3.

The mean duration until sperm was detected (GD 0) varied between 2.2 and 3.6 days.

All sperm positive and negative rats delivered pups or had implants in utero with the following exceptions:
- High-dose females No. 131 - 140 (mated with males No. 31 - 40) did not become pregnant.
- Low-dose female No. 116 (mated with male No. 16) did not become pregnant.

The fertility index was 100% for test groups 1 - 2 and the control and 0% for test group 3. The infertility in the high-dose group (300 mg/kg bw/d) is associated with pathological alterations in the sexual organs (for details see 4.4. Pathology).

The mean duration of gestation was similar in test groups 1 - 2 and the control (i.e. between 22.2 and 22.7 days).

The gestation index varied between 70% (test group 2) and 100% (control and test group 1).

The mean number of implantation sites was 12.3 / 12.7 and 6.6** (**:p≤0.01) implants/dam in test groups 0 - 2 (0, 30 and 100 mg/kg body weight/day). The post-implantation loss was 3.9% / 5.0% and 51.6% in test groups 0 - 2. The mean number of F1 pups delivered per dam was 11.8 / 12.0 and 4.1** (**:p≤0.01) pups/dam in test groups 0 - 2. The changes in the mid-dose group (100 mg/kg bw/d) are considered to be treatment-related.

The rate of liveborn pups was indicated by live birth indices of 100% (test group 2), 98.1% (test group 1) and 99.2% (control).

The number of stillborn pups was comparable between the groups.

Thus, Benzaldehyde, dodecylhydroxy-, oxime, branched (solvent-free and dodecylphenol-depleted) did adversely affect reproduction and delivery of the F0 generation parental females.


ORGAN WEIGHTS (PARENTAL ANIMALS)
Absolute and relative weights

Absolute organ weights
When compared to control group 0 (set to 100%), the mean absolute weights of following organs were significantly changed in one or more test groups (statistically significant changes printed in bold):
See table in any other information on results

All other mean absolute weight parameters did not show significant differences when compared to the control group 0.

Relative organ weights
When compared to control group 0 (set to 100%), the mean relative weights of following organs were significantly changed in one or more test groups (statistically significant changes printed in bold):
See table in any other information on results

The weight changes in reproduction organs, adrenal glands, liver and pituitary gland were regarded to be treatment related.

The terminal body weight was reduced in males of test groups 2 and 3 (100 and 300 mg/kg bw/day) and females of test group 3 (300 mg/kg bw/day). All terminal body weights ranged within the historical control data (see appendix) and were therefore not regarded to be treatment-related. The increase in relative kidney weights of both genders of test group 3 (300 mg/kg bw/day) was regarded to be a consequence to the lower body weight in this group when compared to the control group. It was therefore not regarded to be treatment related.

All other mean relative weight parameters did not show significant differences when compared to the control group 0.

GROSS PATHOLOGY (PARENTAL ANIMALS)

Treatment-related macroscopic findings were observed in males of test groups 2 and 3 and females of test group 3, with incidences according to the table below:
See table in any other information on results
These findings were regarded to be treatment related.

One offspring (pup No. 5) of control female No. 110 showed a dilation of the ductus arteriosus.

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.

Fertility:
All female animals of test group 3 (300 mg/kg bw/day) were not pregnant. This correlated in most cases with the macroscopically observed reduction in ovary size. Many of the male mating partners revealed reduction in organ size of testes, epididymides, prostate and seminal vesicles. These findings were thought to have influenced the fertility in both genders and to be treatment related.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Treatment-related findings were observed in male and females with incidences and grading according to the table below:
See table in any other information on results

Two males of test group 3 (300 mg/kg bw/day) revealed a diffuse degeneration of seminiferous tubules. Seven animals in this group showed a slight reduction in seminiferous tubular diameter. In test group 2 (100 mg/kg bw/day) six animals revealed minimal to slight reduction of the diameter of seminiferous tubules. These findings were regarded to be treatment related.

Four animals of test group 3 (300 mg/kg bw/day) showed a slight to severe reduction in sperm number (oligospermia). In addition, two animals in test group 3 (300 mg/kg bw/day) and one animal of test group 2 (100 mg/kg bw/day) had cellular debris within the tubules. These findings were regarded to be treatment related.

Eight females of test group 3 (300 mg/kg bw/day) and one female of test group 2 (100 mg/kg bw/day) revealed an increased number of atretic follicles. This was characterized by an increase of necrotic and/or apoptotic granulosa cells as well as cellular debris within the follicular lumen. Furthermore, six females of test group 3 (300 mg/kg bw/day) showed an increase of inactive interstitial cells. These animals showed a diminished amount of cytoplasm and condensed nuclei when compared to the control animals. Nine females of (300 mg/kg bw/day) revealed an increased number of cysts and lower numbers of corpora lutea when compared to control females. These findings were regarded to be treatment related.

The macroscopically observed dilation of the ductus arteriosus in pup No. 5 of control female No. 110 was microscopically diagnosed as a dissecting aneurysm of the ductus arteriosus. As it occurred in a control pup, this finding was regarded to be spontaneous.

Fertility
All mating pairs of test group 3 (300 mg/kg bw/day) revealed either findings in the left testicle and/or epididymis or in the ovaries or both. Therefore, the failure in reproduction in all pairs of this test group was regarded to be treatment related.

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.

OTHER FINDINGS (PARENTAL ANIMALS)

CLINICAL PATHOLOGY

Hematology

In rats of both sexes of test group 3 (300 mg/kg bw/d), red blood cell (RBC) counts, hemoglobin and hematocrit values were decreased and relative reticulocyte counts were increased. In females of the mentioned test group mean corpuscular volume (MCV) was decreased and mean corpuscular hemoglobin concentration (MCHC) was increased. Additionally, in females of test group 2 (100 mg/kg bw/d) RBC counts and hematocrit values were already lower compared to controls, but values were within historical control ranges (RBC 7.28-8.25 Tera/L; hematocrit 0.378-0.424 L/L, PART III, Supplement) and therefore these alterations were regarded as incidental and not treatment-related.

In males of test group 3 (300 mg/kg bw/d), prothrombin time (HQT = Hepatoquick’s test) was prolonged.

Some alterations in the differential blood cell counts occurred in rats of both sexes of test groups 2 and 3 (100 and 300 mg/kg bw/d) without any changes of the total white blood cell (WBC) counts, but the values were within historical control ranges: decreased absolute and relative neutrophil counts and absolute monocyte counts in males of test group 3 (300 mg/kg bw/d) and decreased relative neutrophil counts in males of test group 2 (100 mg/kg bw/d); decreased relative eosinophil and basophil counts in females of test group 3 (300 mg/kg bw/d) and increased relative lymphocyte counts in females of test groups 2 and 3 (100 and 300 mg/kg bw/d) (males: absolute neutrophil counts 0.70-1.45 Giga/L; absolute monocyte counts 0.06-0.16 Giga/L; relative neutrophil counts 13.3-25.5 %; females: relative lymphocyte counts 71.1-83.9 %; relative eosinophil counts 1.6-3.1 %; relative basophil counts 0.0-1.3 %, PART III, Supplement). Therefore the changes of the different blood cell counts were regarded as incidental and not treatment-related.

Clinical chemistry

In rats of both sexes of test group 3 (300 mg/kg bw/d), alanine aminotransferase (ALT) activities were increased. Additionally, in females of test group 2 (100 mg/kg bw/d) ALT activities were higher compared to controls, but the values were within the historical control range (ALT 0.43-0.74 µkat/L, PART III, Supplement).

In males of test group 3 (300 mg/kg bw/d) total protein levels were lower compared to controls, which was due to decreased albumin levels. In contrast to that in females of test group 3 (300 mg/kg bw/d) total protein levels were increased which was due to higher globulin levels. Globulin values were already higher in females of test group 2 (100 mg/kg bw/d). However, the values in females were within historical control ranges (total protein 62.13-71.91 g/L; globulins 19.54-31.21 g/L, PART III, Supplement) and in males albumin levels were also within the historical control range whereas total protein levels were marginally below this range (albumin 36.12-40.01g/L, total protein 59.70-69.74 g/L, PART III, Supplement). In females of test group 2 (100 mg/kg bw/d), albumin levels were lower compared to controls, but levels were not dose-dependently changed. Therefore, the altered values of proteins in both sexes were regarded as incidental and not treatment-related.

In rats of both sexes of test groups 2 and 3 (100 and 300 mg/kg bw/d), chloride levels were decreased, but the values were within the historical control ranges apart from the mean of females in test group 3 (males chloride 99.9-107.4 mmol/L, females chloride 100.2-107.8 mmol/L, PART III, Supplement). However, even in females this is the only electrolyte level which was changed and therefore the altered chloride levels were regarded as maybe treatment-related, but not adverse (ECETOC, Technical Report No. 85, 2002).

In females and males of test group 3 (300 mg/kg bw/d), triglyceride levels were higher compared to controls. This was also true for females of test group 2 (100 mg/kg bw/d). However, the values in males of test group 3 as well as in females of test group 2 were within the historical control range (males triglycerides 0.41-1.32 mmol/L, females triglycerides 0.29-0.71 mmol/L, PART III, Supplement). Therefore, at least in males of test group 3 and in females of test group 2 triglyceride changes were regarded as incidental and not treatment-related.

In males of test group 3 (300 mg/kg bw/d), creatinine values were increased and cholesterol values were decreased. In males of test group 2 (100 mg/kg bw/d) cholesterol values were already lower compared to controls. However, in this test group, the decreased cholesterol values was the only changed parameter and therefore, this alteration was regarded as treatment-related, but not adverse (ECETOC, Technical Report No. 85, 2002).

In females of test group 3 (300 mg/kg bw/d), total bilirubin levels were increased.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
general, systemic toxicity
Effect level:
100 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on anemia and altered liver cell metabolism at 300 mg/kg bw/d.
Dose descriptor:
LOAEL
Remarks:
fertility
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Dose-dependent decrease in the number of sperm in the cauda epididymidis as well as a change in relative organ weights of seminal vesicles and pituitary glands were noted at all tested dose levels.
Dose descriptor:
NOAEL
Remarks:
reproductive performance
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
fertility putative
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see discussion for putative NOAEL in the endpoint summary
Remarks on result:
not measured/tested

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
The viability index indicating pup mortality during lactation (PND 0 - 4) varied between 85.7% (test group 2), 98.1% (test group 1) and 99.2% (control). A slightly higher number of decedents (cannibalized/dead) in test group 2 (100 mg/kg bw/d) compared to the control (6 vs. 1) is considered to be treatment-related.

CLINICAL SIGNS (OFFSPRING)
There were no test substance related adverse clinical signs observed in any of the F1 generation pups of the different test groups.

BODY WEIGHT (OFFSPRING)
Mean body weights of the mid-dose (100 mg/kg bw/d) F1 male and female pups were statistically significantly above the concurrent control values on PND 1 (about 14-17%, respectively). Slightly higher mean pup body weights, statistically significant for females, were still noted on PND 4. This is regarded as a secondary effect of the smaller litter size in this group.

Mean pup body weight change of the mid-dose pups was comparable to the concurrent control group during the entire study period.

No test compound-related influence on F1 pup body weights and pup body weight change were noted in test group 1.

One male and one female runt were seen in the control.

GROSS PATHOLOGY (OFFSPRING)
A few pups showed spontaneous findings at gross necropsy, such as post mortem autolysis, dilated ductus arteriosus (control - dam No. 110 male pup No. 5), reddish discolored testis, dilated ureter, and dilated renal pelvis. These findings occurred without any relation to dosing and/or can be found in the historical control data at comparable or even higher incidences (PART III, SUPPLEMENT).

Thus, all these findings were not considered to be associated to the test substance.

OTHER FINDINGS (OFFSPRING)

PUP NUMBER AND STATUS AT DELIVERY
The mean number of delivered F1 pups per dam was statistically significantly reduced in test group 2 (11.8 / 12.0 and 4.1** (**:p≤0.01) pups/dam in test groups 0 - 2).

The rate of liveborn and stillborn F1 pups were evenly distributed about the groups.

The number of cannibalized pups was 1 / 1 and 6 in test groups 0 - 2.

SEX RATIO
The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test group 1.

There was a skew noted in the ratio of live F1 male to female pups in test group 2. The ratios were 33.3% males vs. 66.7% females on PND 0 and and 29.6% males vs. 70.4% females on PND4.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
30 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Fetal and pup mortality at 100 mg/kg bw/d.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Absolute weights

Males

Females

Test group

(mg/kg bw/day)

1

(30)

2

(100)

3

(300)

1

(30)

2

(100)

3

(300)

Terminal body weight

102%

96%*

87%**

99%

94%

88%**

Adrenal glands

104%

110%

151%**

 

 

 

Epididymides

95%

87%**

62%**

 

 

 

Liver

 

 

 

105%

99%

114%*

Ovaries

 

 

 

93%

87%

52%**

Prostate

104%

65%**

35%**

 

 

 

Seminal vesicle

84%*

53%**

16%**

 

 

 

Testes

100%

94%

72%**

 

 

 

*: p <= 0.05, **: p <= 0.01

Relative weights

Males

Females

Test group

(mg/kg bw/day)

1

(30)

2

(100)

3

(300)

1

(30)

2

(100)

3

(300)

Adrenal glands

102%

115%

175%**

104%

1041%

121%**

Epididymides

93%

91%

71%**

 

 

 

Kidneys

101%

102%

110%**

99%

102%

116%**

Liver

102%

107%*

122%**

106%

105%

129%**

Ovaries

 

 

 

94%

93%

54%**

Pituitary gland

117%*

126%**

132%**

105%

102%

126%**

Prostate

102%

68%**

40%**

 

 

 

Seminal vesicle

82%**

56%**

19%**

 

 

 

*: p <= 0.05, **: p <= 0.01

Gross lesions

 

Male animals

Female animals

Test group

(mg/kg bw/day)

0

(0)

1

(30)

2

(100)

3

(300)

0

(0)

1

(30)

2

(100)

3

(300)

No. of animals

10

10

10

10

10

10

10

10

Left epididymis

- size reduced

0

0

0

8

 

 

 

 

Ovaries

- size reduced

 

 

 

 

0

0

0

9

Prostate

- size reduced

0

0

2

9

 

 

 

 

Seminal vesicle

- size reduced

0

0

2

9

 

 

 

 

Left testicle

- size reduced

0

0

0

8

 

 

 

 

Histopathology

Left testis

Male animals

Test group

(mg/kg bw/day)

0

(0)

1

(30)

2

(100)

3

(300)

No. of animals

10

10

10

10

Degeneration tubular diffuse

0

0

0

2

  • Grade 3

 

 

 

2

Tubular size reduced

0

0

6

7

  • Grade 1

 

 

3

 

  • Grade 2

 

 

3

7

Left epididymis

Male animals

Test group

(mg/kg bw/day)

0

(0)

1

(30)

2

(100)

3

(300)

No. of animals

10

10

10

10

Oligospermia

0

0

0

4

  • Grade 2

 

 

 

1

  • Grade 3

 

 

 

1

  • Grade 5

 

 

 

2

Debris in seminiferous tubules

0

0

1

3

  • Grade 1

 

 

1

1

  • Grade 2

 

 

 

2

Ovaries

Female animals

Test group

(mg/kg bw/day)

0

(0)

1

(30)

2

(100)

3

(300)

No. of animals

10

10

10

10

Follicles, atretic, increased

0

0

1

8

  • Grade 1

 

 

 

3

  • Grade 2

 

 

 

4

  • Grade 3

 

 

1

1

Inactivity, interstitial cells

0

0

0

6

Cyst(s)

1

0

0

9

Corpora lutea, decreased

0

0

1

9

The sperm head counts in the cauda epididymidis were (TS/gT [Mio/g] on day 50):

0 mg/kg bw/d: Mean:486

30 mg/kg bw/d: Mean:422*

100 mg/kg bw/d: Mean:372**

300 mg/kg bw/d: Mean:252**

*: p<= 0.05; **: p<= 0.01

Applicant's summary and conclusion

Conclusions:
Under the conditions of this OECD 422 combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in Wistar rats the following NOAEL (no observed adverse effect level) of Benzaldehyde, dodecylhydroxy-, oxime, branched (solvent-free and dodecylphenol-depleted) were determined:

The NOAEL for general, systemic toxicity was 100 mg/kg bw/d for the F0 males and females based on anemia and altered liver cell metabolism at 300 mg/kg bw/d.

No NOAEL for fertility was determined for the F0 parental males because a dose-dependent decrease in the number of sperm in the cauda epididymidis as well as a change in relative organ weights of seminal vesicles and pituitary glands were noted at all tested dose levels of 30 mg/kg bw/d and above. Fertility and reproductive performance of F0 parental females were unaffected at 30 mg/kg bw/d. Fertility and reproductive performance were distinctly impaired at 100 mg/kg bw/d, while outright infertility was the result of the exposure to 300 mg/kg bw/d.

The NOAEL for developmental toxicity in the F1 offspring was 30 mg/kg bw/d, based on fetal and pup mortality at 100 mg/kg bw/d.