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Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Based on an OECD Guideline 422 and GLP study with diisopropanol-p-toluidin, the NOAEL for reproductive toxicity was considered to be 20 mg/kg bw/day for all relevant endpoints, which was the highest dose tested in female animals.

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: combined repeated dose and reproduction/developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-05-22 to 2013-02-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented GLP and guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA, OPPTS 870.3650 (Combined Repeated dose toxicity study with the reproduction/developmental toxicity screening test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
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: 10 - 11 weeks old
- Housing: 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 (end of lactation). For motor activity (MA) measurements the animals were housed individually in polycarbonate cages with small amounts of bedding material. Pregnant females were provided with nesting material (cellulose wadding) toward the end of gestation.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 - 24°C
- Humidity: 30 - 70%
- Air changes: 15 air changes per hour
- Photoperiod: The day/night cycle was 12 hours.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
To prepare this solution, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, corn oil was filled up to the desired volume, subsequently released for 30-40 minutes in an ultrasonic bath. The test substance preparations were produced at least once a week. After the reduction of the dose level of the female animals from 40 mg/kg bw/d to 20 mg/kg bw/d the test substance preparations were produced for the first study week as follows:
30 mL of the high dose preparation (40 mg/kg bw/d) was filled up with 30 mL of corn oil to obtain the desired dose of 20 mg/kg bw/d.

VEHICLE
- Justification for use and choice of vehicle:
Corn oil is a standard vehicle for studies of this type.
- Concentration in vehicle:
0.625, 2.5, 5, 10 mg/L
Details on mating procedure:
MATING OF F0 GENERATION PARENTAL ANIMALS
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 test group. 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 was detected was denoted gestation day (GD) 0 and the following day GD 1.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in corn oil for a period of 7 days at room temperature was proven before the start of the study. Concentration control analysis of the test substance preparations were performed in samples of all concentrations at the start of the administration period. Given that the test substance is completely miscible with corn oil, solutions were considered to be homogenous without further analysis.
Duration of treatment / exposure:
The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation and two weeks thereafter in females.
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0,2.5, 10 (both sexes), 20 [40 on pre-mating day 0] mg/kg bw/d (females only) and 40 mg/kg bw/d (male only).
Basis:

No. of animals per sex per dose:
10 male and 10 female animals
Control animals:
yes, concurrent vehicle
Positive control:
no
Parental animals: Observations and examinations:
MORTALITY: Yes
- Time schedule: A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.


CLINICAL OBSERVATIONS: Yes
- Time schedule: 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 affected animal.The littering and lactation behavior of the dams was generally evaluated in the mornings in combination with the daily clinical inspection of the dams. On weekdays (except public holidays) the parturition behavior of the dams was inspected in the afternoons in addition to the evaluations in the mornings.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:Detailed clinical observations (DCO) were performed in all animals prior to the administration period and thereafter at weekly intervals. The following parameters were examined:

1. abnormal behavior during “handling”
2. fur
3. skin
4. posture
5. salivation
6. respiration
7. activity/arousal level
8. tremors
9. convulsions
10. abnormal movements
11. impairment of gait
12. lacrimation
13. palpebral closure
14. exophthalmus
15. feces (appearance/consistency)
16. urine
17. pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was determined before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning). The following exceptions are notable for the female animals:
1. 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.
2. Females with litter were weighed on the day of parturition (PND 1) and on PND 4.
3. Females without a litter and without positive evidence of sperm in the vaginal smear 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:
1. Food consumption was not determined during the mating period (male and female F0 animals).
2. Food consumption of the F0 females with evidence of sperm was determined on GD 0 - 7, 7 - 14 and 14 - 20.
3. Food consumption of F0 females, which gave birth to a litter was determined for PND 1 - 4.

Food consumption was not determined in females without positive evidence of sperm (during the mating period of dams used in parallel) and females without litter (during the lactation period of dams used in parallel) and in males after the premating period.

WATER CONSUMPTION: Yes
Drinking water consumption was monitored by daily visual inspection of the water bottles for any changes in volume.

HEMATOLOGY: Yes
- Time schedule for collection of blood: In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The following parameters were determined in blood:
- Leukocyte count (WBC),
- Erythrocyte count (RBC),
- Hemoglobin (HGB),
- Hematocrit (HCT),
- Mean corpuscular volume (MCV),
- Mean corpuscular hemoglobin (MCH),
- Mean corpuscular hemoglobin concentration (MCHC),
- Platelet count (PLT),
- Differential blood count,
- Reticulocytes (RET),
- Prothrombin time (Hepato Quick’s test) (HQT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: please refer to hematology
The following clinicochemical parameters were determined:
- Alanine aminotransferase (ALT),
- Aspartate aminotransferase (AST),
- Alkaline phosphatase (ALP),
- γ-Glutamyltransferase (GGT),
- Sodium (NA),
- Potassium (K),
- Chloride (CL),
- Inorganic phosphate (INP),
- Calcium (CA),
- Urea (UREA),
- Creatinine (CREA),
- Glucose (GLUC),
- Total bilirubin (TBIL),
- Albumin (ALB),
- Globulins (GLOB),
- Triglycerides (TRIG),
- Cholesterol (CHOL),
- Bile acids (TBA).

URINALYSIS: Yes
- Time schedule for collection of urine: For urinalysis the individual animals were transferred to metabolism cages (withdrawal of food and water) and urine was collected overnight. Urine samples were evaluated in a randomized sequence.
The following parameters were determined:
- pH,
- Protein,
- Glucose,
- Ketones,
- Urobilinogen,
- Bilirubin,
- Blood,
- Specific gravity,
- Sediment,
- Color, turbidity,
- Volume.

NEUROBEHAVIOURAL EXAMINATION: Yes
1. Functional observation battery
A functional observational battery was performed in the first five male animals per test group and the first 5 female animals with litter of all test groups at the end of the administration period. The FOB started 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.

Home cage observation:
1. Posture
2. Tremors
3. Convulsions
4. Abnormal movements
5. Impairment of gait

Open field observation:
1. Behavior when removed from cage
2. Fur
3. Skin
4. Salivation
5. Nose discharge
6. Lacrimation
7. Eyes/ pupilsize
8. Posture
9. Palpebral closure
10. Respiration
11. Tremors
12. Convulsions
13. Abnormal movements/ stereotypes
14. Impairment of gait
15. Activity/ arousal level
16. Feces excreted within 2 minutes (number/ appearance/ consistency)
17. Urine excreted within 2 minutes (amount/ color)
18. Rearing within 2 minutes

Sensory motor tests/reflexes:
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 (Auditory startle response)
7. Coordination of movements (Righting response)
8. Behavior during handling
9. Vocalization
10. Pain perception (Tail pinch)
11. Grip strength of forelimbs
12. Grip strength of hindlimbs
13. Landing foot-splay test
14. Other findings

2. Motor activity assessment
Motor activity (MA) was also measured on the same day as the FOB was performed in the first five parental males and females (with litter) per group.
Litter observations:
Litter/Pups
- Pup number and status at delivery
- Sex ratio
- Pup clinical observations
- Pup body weight data
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 males sacrificed on schedule:
1. Anesthetized animals
2. Epididymides
3. Testes
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
2. Brain
3. Heart
4. Kidneys
5. Liver
6. Spleen
7. Thymus

HISTOPATHOLOGY
The following organs were examined:
- Adrenal glands,
- All gross lesions,
- Bone marrow (femur),
- Brain,
- Cecum,
- Cervix,
- Coagulating glands,
- Colon,
- Duodenum,
- Epididymides,
- Heart,
- Ileum,
- Jejunum,
- Kidneys,
- Liver,
- Lungs,
- Lymph nodes (axillary and mesenteric),
- Ovaries,
- Oviducts,
- Prostate gland,
- Peyer’s patches,
- Rectum,
- Sciatic nerve,
- Seminal vesicles,
- Spinal cord (cervical, thoracic, lumbar),
- Spleen,
- Stomach (forestomach and glandular stomach),
- Testes,
- Thymus,
- Thyroid glands,
- Trachea,
- Urinary bladder,
- Uterus,
- Vagina.
Postmortem examinations (offspring):
- Pup necropsy observations
Statistics:
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, implantation sites, pups delivered, life pups day:
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 delivering, 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:
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.

Viability 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.

Feces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity:
Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pair-wise comparison of each dose group with the control group was performed using WILCOXON test (two-sided) for the hypothesis of equal medians.

Statistics of clinical pathology and pathology:
Please refer to any other information on material and methods incl. tables
Reproductive indices:
Male reproduction data:
- male mating index (%)
- male fertility index (%)

Female reproduction and delivery data:
- female mating index (%)
- female fertility index (%)
- gestation index (%)
- live birth index (%)
Offspring viability indices:
- Pup viability/mortality (Viability index (%))
MORTALITY
Three female animals of the high dose group were found dead when treated with 40 mg/kg bw/d on premating study day 1. Following reduction of the dose level for high dose group females from 40 to 20 mg/kg bw/day, there were no further incidences of mortality.

DETAILED CLINICAL OBSERVATION

Summary of clinical obserevations for males and females:
- Nine female animals of the 40 mg/kg bw/d dose group showed severe tremors on premating day 1. Three females of the 40 mg/kg bw/d dose group were found dead on premating day 1. In response, the dose level was reduced to 20 mg/kg bw/d in female animals for the remainder of the treatment period.
- Three male animals (40 mg/kg bw/d), one female animal (20 [40 on pre-mating day 0] mg/kg bw/d) and two female animals (10 mg/kg bw/d) showed salivation during the premating period.
- One male animal (2.5 mg/kg bw/d), four male animals (40 mg/kg bw/d) and one female animal (10 mg/kg bw/d) showed salivation during the mating period.
- Two male animals (40 mg/kg bw/d) showed salivation during the postmating period.

The salivation in different test groups on different time points in both sexes was assessed as substance-related but not as an adverse effect, due to the taste of the substance.

Summary of clinical obserevations for females during gestation:
One female animal (10 mg/kg bw/d) showed slight salivation. This finding was assessed as substance-related but not as an adverse effect, due to the taste of the substance.
Clinical observation for females during lactation:
No clinical signs were observed.

BODY WEIGHT DATA
No deviations in the body weights of all test groups were observed.

FOOD CONSUMPTION
Female animals (10 mg/kg bw/d) showed increased food consumption values on gestation day 7 (+8.2%) compared to the control group.
No other significant deviations in male and female animals were observed during the study period; therefore no dose response relationship was observed and this finding was assessed as being incidental.

WATER CONSUMPTION
No test substance-related findings were noted.

HEMATOLOGY
No treatment-related changes among hematological parameters were observed. In males of the 2.5 and 10 mg/kg bw/d dose group mean corpuscular volume (MCV) was increased and additionally in males of the 2.5 mg/kg bw/d dose group, mean corpuscular hemoglobin content (MCH) was higher compared to controls. Both calculated parameters were not dose-dependently changed and the measured red blood cell parameters (i.e., red blood cell (RBC) counts, hemoglobin concentration and hematocrit) were not altered in these animals. Therefore, these changes were regarded as incidental and not treatment-related.

CLINICAL CHEMISTRY
No treatment-related changes among clinical chemistry parameters were observed.

URINALYSIS
No treatment-related changes among urinalysis parameters were observed.

NEUROBEHAVIOUR
- Home cage observations: No test substance-related effects were observed.
- Open field observations: No test substance-related effects were observed.
- Sensorimotor rests/reflexes: No test substance-related effects were observed.
- Quantitive parameters: No test substance-related effects were observed.
- Motor activity measurement: There were no significant deviations concerning the overall motor activity (summation of all intervals) in male and female animals.

ORGAN WEIGHTS
When compared to the control group (set to 100%), the mean absolute liver weights were significantly increased in male animals of the 2.5 mg/kg bw/d dose group and of the 40 mg/kg bw/d dose group. All other mean absolute weight parameters in males and all weight parameters in females did not show significant differences when compared to the control group. None of the mean relative weight parameters showed significant differences when compared to the control group. Absolute liver weights of male animals did not show a dose response, there were no significant changes in relative liver weights, and a histopathological correlate was not detected, therefore increased absolute liver weights were regarded as incidental.

GROSS LESIONS
All findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment. None of the animals premature decents showed any gross lesions.

GROSS LESIONS - FERTILITY
Two mating pairs of the 2.5 mg/kg bw/d dose group, one pair of the 10 mg/kg bw/d dose group and three pairs of the 40/20 mg/kg bw/d dose group were recorded as no offspring/ not pregnant.
Mating pairs of the 2.5 mg/kg bw/d dose group and of the 10 mg/kg bw/d dose group did not show gross lesions. Two male animals of the 40 mg/kg bw/d dose group showed relevant macroscopic lesions: A focus was observed on the right epididymis of a male animal, and the organ size of the left epididymidis was reduced in other male animal.

HISTOPATHOLOGY
All findings occurred either individually or were 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 relevant histopathological findings in any of the animals having died spontaneously.
HISTOPATHOLOGY - FERTILITY
Two mating pairs of the 2.5 mg/kg bw/d dose group, one pair of the 10 mg/kg bw/d dose group and three pairs of the 40/20 mg/kg bw/d dose group were recorded as no offspring/ not pregnant.
Among these mating pairs, two male animals (Nos. 32 and 39 ) of the 40 mg/kg bw/d dose group both showed a unilateral sperm granuloma in the epididymis correlating to “focus” macroscopically in animal No. 32. Male animal No. 39 showed additionally immature (not dilated, empty) ducts of the contralateral epididymis, correlating to reduced size of this organ at necropsy. The testis of that side was missing in animal No. 39.

MALE REPRODUCTION DATA
- Male mating index: The male mating index was 100% in all test groups.
- Male fertility index: Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter. One male of the 10 mg/kg bw/d dose group and 2 males of the 2.5 mg/kg bw/d dose group did not generate F1 pups. The male fertility index was 80% in the 2.5 mg/kg bw/d dose group, 90% in the 10 mg/kg bw/d dose group and 100% in the 40 mg/kg bw/d dose group and the control group. This finding reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

FEMALE REPRODUCTION AND DELIVERY DATA
- Female mating index: The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm was detected (GD 0) was 3.1, 2.5, 1.8 and 1.6 days in the control, low, mid and high dose groups, respectively. This finding reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
- Female fertility index: The female fertility index was 80% in the 2.5 mg/kg bw/d, 90% in the 10 mg/kg bw/d and 100% in the 20 [40 on pre-mating day 0] mg/kg bw/d dose group and the control group. Female animals , which delivered no pups, showed no implantation sites. The mean duration of gestation was similar in all test groups. This finding reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
- Gestation index: The gestation index was 100% in all test groups. Live birth indices: The rate of liveborn pups was not affected by the test substance, as indicated by live birth indices of 99% - 100% in all test groups.
- Postimplantation loss: The postimplantation loss was 3.38 % in the control group, 0.96 % in the 2.5 mg/kg bw/d dose group, 6.62 % in the 10 mg/kg bw/d dose group and 1.02 % in the 20 [40 on pre-mating day 0] mg/kg bw/d dose group. These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
Dose descriptor:
NOAEL
Remarks:
(systemic toxicity)
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: There were no signs of systemic toxicity at the highest dose level of 40 mg/kg bw/day in male parental animals.
Dose descriptor:
NOAEL
Remarks:
(systemic toxicity)
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Remarks:
(reproductive performance and fertility)
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No reproduction effects were noted up to the highest dose tested in male animals.
Dose descriptor:
NOAEL
Remarks:
(reproductive performance and fertility)
Effect level:
20 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No reproduction effects were noted up to the highest dose tested in female animals.
F1 GENERATION LITTER/PUPS

- Pup number and status at delivery:The mean number of delivered F1 pups per dam was evenly distributed among the groups.

- Pup viability/mortality: The viability index indicating pup mortality during lactation (PND 0 - 4) was 98.7% (20 [40 on pre-mating day 0] mg/kg bw/d dose group), 99.1% (10 mg/kg bw/d dose group) 100% (2.5 mg/kg bw/d dose group and control group) and was in the normal range of biological variation inherent in the strain of rats used for this study. One female pup of control group and 10 mg/kg bw/d dose group were found stillborn. These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
- 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 substance-treated groups; slight differences were regarded to be spontaneous in nature.
- Pup clinical observations: The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4.
- Pup body weight data: Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group. Four male runts were seen in the 2.5 mg/kg bw/d dose group. These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
- Pup necropsy observation: One F1 pup of the 2.5 mg/kg bw/d dose group showed post mortem autolysis and one pup of the 20 mg/kg bw/d dose group was cannibalized. These findings were assessed as being spontaneous in nature and without biological relevance.
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test substance-related, advers findings were observed
Remarks on result:
other: highest tested dose
Reproductive effects observed:
not specified

DOSE FORMULATION ANALYSIS

The stability of the test substance in corn oil was demonstrated over a period of 7 days at room temperature. The analytically determined test substance concentrations in the vehicle were in the expected range of the nominal concentrations (91.5 -103.2%) for all formulations.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Combined Repeated dose Toxicity study with the Reproduction/Developmental Toxicity Screening Test: oral route  

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (BASF SE, 2013, OECD 422) diisopropanol-p-toluidin was administered to Wistar rats (4 groups of 10 male and 10 female animals each) via oral gavage at dose levels of 0, 2.5, 10, 40 mg/kg bw/day (males) and 20 [40 on pre-mating day 0] mg/kg bw/day (females). The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation and two weeks thereafter in females. Mortality, clinical signs, body weight, food and water consumption were assessed at regular intervals. At the end of the study functional observation battery and motor activity parameters were determined. Clinicochemical and hematological examinations as well as urinalyses were performed in 5 animals per sex and group towards the end of the administration period. At necropsy, selected organs were weighed and the animals were examined macroscopically and histopathologically. Relevant reproductive parameters and indices were determined. The pups were sexed,weighed and examined for macroscopically evident changes. Their viability was recorded. At necropsy, all pups were sacrificed under isoflurane anesthesia with CO2 and examined macroscopically for external and visceral findings.  

No mortality and no clinical signs were observed for male animals. Three female animals of the high dose group (40 mg/kg bw/d) were found dead on premating day 1. In addition, nine females showed severe tremors on premating day 1. After reduction of the dose level to 20 mg/kg bw in female animals no signs of general systemic toxicity were observed. No treatment-related effects were observed in mean body weight, body weight changes, food and water consumption of exposed animals throughout the study. Neurological testing did not indicate any neurotoxic potential of the test item. No treatment related changes among hematological, clinical chemistry and urinalysis parameters were observed. When compared to the control (set up 100%), the mean absolute liver weights were significantly increased in male animals of the 2.5 and 40 mg/kg bw/d dose group. Absolute liver weights of male animals did not show a dose response, there were no significant changes in relative liver weights, and a histopathological correlate was not detected, therefore increased absolute liver weights were regarded as incidental. All other mean absolute weight parameters in males and all weight parameters in females did not show significant differences when compared to the control group. At gross lesions and histopathology examination all findings were considered to be incidental or were biologically equally distributed over control and treatment groups. Parameters for reproductive performance i.e. male and female mating and fertility index, gestation index and postimplantation loss revealed no test substance-related adverse findings. Two mating pairs of the low dose group, one pair of the mid dose group and three pairs high dose group were recorded as not pregnant. Thereby, two male animals of the high dose group showed gross lesions and histopathological findings. The findings were considered to be incidental or spontaneous in origin and without relation to treatment. For F1 pups, viability/mortality, sex ratio, clinical observation, body weight data and necropsy examinations indicated no adverse findings and were assessed as being spontaneous in nature and without biological relevance. In conclusion, based on mortality and tremors noted at 40 mg/kg bw/d the No Observed Adverse Effect Level (NOAEL) for females was established at 20 mg/kg bw/day. As no systemic toxicity was observed in males the NOAEL was established at 40 mg/kg bw/d. Due to the lack of any relevant findings the NOAEL for reproductive performance and fertility as well as the NOAEL for developmental toxicity in the F1 progeny was considered to be 40 mg/kg bw/d in males and 20 mg/kg bw/d in females.

Effects on developmental toxicity

Description of key information
In an OECD Guideline 414 and GLP study with diisopropanol-p-toluidin in rats, the NOAEL for maternal and prenatal developmental toxicity was 20 mg/kg/day, which was the highest dose tested in female animals.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 2017 - August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
Time-mated Wistar rats (Crl:WI[Han]) were supplied by Charles River Laboratories, Research Models and Services, Germany GmbH, at an age of about 10-12 weeks. Only animals free from clinical signs of disease were used for the investigations.

During the study period, the rats were housed individually in Polycarbonate cages type III supplied by TECNIPLAST, Hohenpeißenberg, Germany and Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm²). Individual housing is preferred over group housing as the close individual monitoring of food and water consumption as well as of clinical signs of toxicity in pregnant animals is crucial during this period of increased sensitivity. In addition, the control for signs of abortion or fetal loss can only be done in a reliable fashion with single-housed animals.
Dust-free wooden bedding was used in this study. For enrichment, wooden gnawing blocks were offered (Typ Lignocel® block large, supplied by J. Rettenmaier & Söhne GmbH + Co KG, Rosenberg, Germany). The animals were accommodated in fully air-conditioned rooms in which central air conditioning maintained a range of temperature of 20-24°C and a range of relative humidity of 30-70%. The air exchange rate was 15 times per hour. There were no deviations from these limits during the entire study. 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). The food used was ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland. Food and drinking water (potable tap water in water bottles)
were available ad libitum.
Route of administration:
oral: gavage
Details on exposure:
The test substance preparations were administered to the animals once a day orally by gavage, from implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always at approximately the same time in the morning. The animals of the control group were treated with the vehicle (corn oil) in the same way. The volume administered each day was 4 mL/kg body weight. The calculation of the administration volume was based on the most recent individual body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The results of the analysis of the test substance preparations confirmed the correctness of the prepared concentrations. The measured concentrations of the samples corresponded to the expected values within the limits of the analytical method, i.e. were always above 90% and below 110% of the nominal concentrations
Details on mating procedure:
The animals were paired by the breeder (“time-mated”); the day of evidence of mating (= detection of vaginal plug/sperm) was referred to as GD 0. The animals arrived on the same day (GD 0) at the experimental laboratory.
Duration of treatment / exposure:
The test substance preparations were administered to the animals once a day orally by gavage, from implantation to one day prior to the expected day of parturition (GD 6 to GD 19), always at approximately the same time in the morning.
Frequency of treatment:
once a day
Duration of test:
On GD 20, the females were sacrificed in a randomized order and examined macroscopically.
Dose / conc.:
2.5 mg/kg bw/day (nominal)
Dose / conc.:
7 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (nominal)
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
DOSE RATIONALE:
In a reproduction/developmental toxicity screening test (OECD 422), Diisopropanol-p-toluidin was administered orally via gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg bw/d (test group 0), 2.5 mg/kg bw/d (test group 1), 10 mg/kg bw/d (test group 2) and 40 (males)/ 20 (females) mg/kg bw/d (test group 3). Almost all (9 out of 10) females of test group 3 (40 mg/kg bw/d) showed severe tremors on premating day 1 and three females (40 mg/kg bw/d) were found dead on premating day 1. After the death of these animals receiving 40 mg/kg bw/d, the dose level was reduced to 20 mg/kg bw/d (only in females) from study day 2 onwards. No further findings were observed.

Based on the lethal / severe findings in the OECD 422 and at the request of the sponsor, the following dose levels were chosen for the present prenatal developmental toxicity study in Wistar rats:
- 2.5 mg/kg body weight/day: as low-dose level
- 7 mg/kg body weight/day: as mid-dose level
- 20 mg/kg body weight/day: as high-dose level

The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.
Maternal examinations:
Mortality
A check was made twice a day on working days or once a day on Saturdays, Sundays or on public holidays (GD 0-20).

Clinical symptoms
A cage-side examination was conducted at least once daily before and after treatment period (GD 0-5 and 20). During treatment period (GD 6-19) all rats were checked daily for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity before administration as well as within 2 hours and within 5 hours after administration.

Food consumption
The consumption of food was recorded for the intervals GD 0-1, 1-3, 3-6, 6-8, 8-10, 10-13, 13-15, 15-17, 17-19 and 19-20.

Body weight data
All animals were weighed on GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20. The body weight change of the animals was calculated based on the obtained results

Corrected (net) body weight gain
Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 20 minus weight of the unopened uterus minus body weight on GD 6).
Ovaries and uterine content:
On GD 20, the dams were sacrificed under isoflurane anesthesia by decapitation, in
randomized order.
After the dams had been sacrificed, they were necropsied and assessed for gross pathology.
The uteri and the ovaries were removed and the following data were recorded:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
• Live fetuses
• Dead implantations:
a) Early resorptions (only decidual or placental tissues visible or according to SALEWSKI
from uteri from apparently non-pregnant animals and the empty uterus horn in the
case of single horn pregnancy)
b) Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c) Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the
uterus had been opened)
After the weight of the uterus had been determined, all subsequent evaluations of the dams
and the gestational parameters (except of gross pathology including organ weights) were conducted
by technicians unaware of treatment group in order to minimize bias. For this purpose
animal numbers were encoded.

These data were used to calculate conception rate and pre- and postimplantation losses.
Fetal examinations:
At necropsy each fetus was weighed, sexed, and external tissues and all orifices were examined macroscopically. The sex was determined by observing the distance between the anus
and the base of the genitalia. Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. The placentas were weighed and their individual weights were recorded.
Thereafter, the fetuses were sacrificed by a subcutaneous injection of pentobarbital (Narcoren®; dose: 0.1 mL/fetus). After these examinations, approximately one half of the fetuses per dam were eviscerated, skinned and fixed in ethanol; the other half was placed in Harrison’s fluid for fixation.

Soft tissue examination of the fetuses
The fetuses fixed in Harrison’s fluid were examined for any visceral findings according to the method of BARROW and TAYLOR. After this examination these fetuses were discarded.

Skeletal examination of the fetuses
The skeletons of the fetuses fixed in ethanol were stained according to a modified method of KIMMEL and TRAMMELL. Thereafter, the skeletons of these fetuses were examined under a stereomicroscope. After this examination the stained fetal skeletons were retained individually.
Statistics:
see attached background material
Historical control data:
see attached background material
Clinical signs:
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 female at dose levels of 2.5, 7 or 20 mg/kg bw/d during the entire study period.
For one dam of the low-dose group (No. 36 - 2.5 mg/kg bw/d) vaginal hemorrhage before and after treatment (>2h<5h) was recorded on GD 19. Since it was only one affected animal and there was no relation to dose, it was not assessed as treatment-related.

Please see also attached summary document.
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any females of all test groups (0, 2.5, 7 or 20 mg/kg bw/d).
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The mean body weights and the average body weight gain of the low-, mid- and high-dose dams (2.5, 7 and 20 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period.
The statistically significantly increased body weight gain value in test group 3 during GD 19-20 was assessed as incidental.

Please see also attached summary document.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption of the high-, mid- and low-dose dams (20, 7 and 2.5 mg/kg bw/d) was generally comparable to the concurrent control group throughout the entire study period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Uterus weight
The mean gravid uterus weights of the animals of test groups 1-3 (2.5, 7 and 20 mg/kg bw/d) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.

Weight of the placentae
The mean placental weights of test groups 1-3 were comparable to the concurrent control group.
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Two spontaneous findings were noted in two individual females of test group 3 (20 mg/kg bw/d), i.e. dilated renal pelvis (No. 77) and discolored kidneys (No. 78). No necropsy findings which could be attributed to the test substance were seen in any dam.

Please see also attached summary document.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Reproduction data
The conception rate was 96% in the control and the mid-dose groups (0 and 7 mg/kg bw/d) and 100% in the low- and high-dose groups (2.5 and 20 mg/kg bw/d). With these rates, a sufficient number of pregnant females were available for the purpose of this study. There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for the pre- and post-implantation losses, the number of resorptions and viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.

For historical control data see also attached background material.
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
not specified
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No treatment-related, advers effects were observed
Remarks on result:
other: highest tested dose
Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the concurrent control group.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
The sex distribution of the fetuses in test groups 1-3 (2.5, 7 and 20 mg/kg bw/d) was comparable to the control fetuses.
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
External malformations were detected in test groups 1 (kinked tail) and 2 (multiple external malformations) (2.5 and 7 mg/kg bw/d). In one case, these external malformations were associated with skeletal malformations. None of these malformations were assessed as treatment-related since they were not related to dose and the finding multiple external malformations can be found in the historical control data at comparable incidences.
The total incidence of external malformations in treated animals did not differ significantly from the concurrent control group and was covered by the historical control data.

For historical control data see also attached background material.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Skeletal malformations were noted in one fetus, each, in test groups 1 (cleft sternum) and 2 (multiple skeletal malformations) (2.5 and 7 mg/kg bw/d). One male fetus of the mid-dose group had multiple skeletal findings concerning skull, forelimbs, sternum and vertebral column. The incidences of the total skeletal malformations were not dose-dependent. The malformations were not assessed as treatment-related since they occurred in single fetuses without relation to dose and were within the range of the respective historical control data.

For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dose. The overall incidences of skeletal variations were comparable to the historical control data.

The increased incidences of skeletal variations were not related to the dose and/or they were inside the historical control range (see attached background material). Therefore, they are not considered as treatment-related.

For historical control data see also attached background material.
Visceral malformations:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No treatment-related, advers effects observed
Remarks on result:
other: highest tested dose
Key result
Developmental effects observed:
no
Conclusions:
In a prenatal developmental toxicity study, the test substance Diisopropanol-p-toluidin was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity.
Analyses confirmed the correctness of the prepared concentrations, the homogeneous distribution and the stability of the test substance in the vehicle. Neither clinical examinations nor determination of food consumption and body weights revealed any relevant difference between animals receiving 2.5, 7 or 20 mg/kg bw/d Diisopropanol-p-toluidin and the control. No differences of toxicological relevance between the control and the treated groups were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no influence of the test substance on fetal weight and sex distribution of the fetuses was noted at any dose.

Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose.
Executive summary:

Under the conditions of this prenatal developmental toxicity study, the oral administration of Diisopropanol-p-toluidin to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6-19) at doses as high as 20 mg/kg bw/d caused neither evidence of maternal nor developmental toxicity.

In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is the highest tested dose of 20 mg/kg bw/d.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
20 mg/kg bw/day
Study duration:
subacute
Species:
rat
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Prenatal Developmental Toxicity Study in Wistar Rats Oral Administration (Gavage)

In a prenatal developmental toxicity study, the test substance diisopropanol-p-toluidin was administered to pregnant Wistar rats daily by gavage from implantation to one day prior to the expected day of parturition (GD 6-19) to evaluate its potential maternal and prenatal developmental toxicity.

Neither clinical examinations nor determination of food consumption and body weights revealed any relevant difference between animals receiving 2.5, 7 or 20 mg/kg bw/d diisopropanol-p-toluidin and the control. No differences of toxicological relevance between the control and the treated groups were determined for any reproductive parameters, such as conception rate, mean number of corpora

lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no influence of the test substance on fetal weight and sex distribution of the fetuses was noted at any dose.

Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose.

Justification for classification or non-classification

Based on the results obtained from reproduction/developmental testing (OECD 422, OECD 414), the test substance is not considered to be subject to classification and labelling for toxicity to reproduction/development according to Regulation (EC) No 1272/2008 (CLP).