Bis(4-(1,1,3,3-tetramethylbutyl)phenyl)amine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproductive Toxicity Study:

In the combined repeated-dose/reproductive/developmental toxicity screening study, the test material was administered by gavage to three groups each of ten male and ten female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for up to fifty-four consecutive days, at dose levels of 50, 250 and 600 mg/kg/day. The following was recorded for each female: Date of mating, Date and time of observed start of parturition, Date and time of observed completion of parturitionand Duration of gestation. After dosing, it was observed that,Females treated with 600 mg/kg/day showed a higher percentage of pre-implantation losses in comparison to controls, resulting in the birth of less offspring per litter and lower total litter weights at this dose level. Thus, no adverse effects (NOAEL) on mating performance, fertility or gestation were observed at 250 mg/kg-bw/day of concentartion. Also, the same NOAEL was considered for fetal parameters. Thus, based on all the observation and results, it was concluded that the NOAEL of the test chemical was 250 mg/kg bw after administration to the test animals.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Justification for type of information:

Weight of evidence based on the data of test chemicals.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

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

GLP compliance:

yes

Limit test:

no

Justification for study design:

No Data Available

Species:

other: Study 2: rat Study 3: rat Study 4: rat; Study 5: rat

Strain:

other: Study 2: Sprague-Dawley Study 3: Sprague-Dawley Crl:CD· (SD) IGS DR Strain Rat Study 4: Slonaker-Addis; Study 5: Crj: CD(SD)

Details on species / strain selection:

No Data Available

Sex:

male/female

Details on test animals or test system and environmental conditions:

Study 2: No Data Available

Study 3: TEST ANIMALS
- Source: Charles River Limited. (UM) , Margate, kent .
- Age at study initiation: Approximately 8 weeks
- Weight at study initiation: Parental males weighed 185-230g and parental females weighed 150-187g.
- Housing: Animals were housed in groups of five in polypropylene cages with stainless steel grid floors and tops, suspended over polypropylene trays lined with absorbent paper. Mated females were housed individually during gestation and lactation, in polypropylene cages with
solid floors and stainless steel lids, furnished with softwood flakes.
- Diet (e.g. ad libitum): Rodent PMI 5002 pelleted diet, ad libitum
- Water (e.g. ad libitum): drinking water was supplied from polycarbonate bottles attached to the cage, Ad libitum
- Acclimation period: At least 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 2 1±2°C
- Humidity (%): 55 ±15%
- Air changes (per hr): At least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hr dark/12-hr light

Other details :
The animals were allocated to dose groups using a randomization procedure based on stratified bodyweights and the group mean body weights were then determined to ensure similarity between the dose groups. The animals were uniquely identified within the study, by an ear punching system.

Study 4: - Age : 5 weeks old
-Diet (e.g. ad libitum) : The Addis diet,ad libitum
-Water (e.g. ad libitum): Ad libitum

Study 5: Details on test animal
TEST ANIMALS
- Source: No data available
- Age at study initiation: (P) x wks; (F1) x wks: P- 9 weeks old
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g: No data available

- Fasting period before study: No data available
- Housing: No data available
- Diet (e.g. ad libitum): No data available
- Water (e.g. ad libitum): No data available
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data available
- Humidity (%):No data available
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): No data available

Route of administration:

other: Study 2: oral: gavage; Study 3: oral: gavage; Study 4: oral: feed; Study 5: oral: gavage

Vehicle:

other: Study 2: corn oil; Study 3: not specified; Study 4: Basal diet; Study 5: olive oil

Details on exposure:

Study 2: The test material was administered by gavage to three groups each of ten male and ten female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for up to fifty-four consecutive days, at dose levels of 50, 250 and 600 mg/kg/day.A control group of ten males and ten females was dosed with vehicle alone (corn oil).
Exposure period: Males: 43 days; Females: up to 54 days

Study 3: 48 consecutive days

Study 4: The test chemical was mixed into the basal diet of commercial laboratory chow at concentrations of 0.0 (vehicle), 67 mg/kg bw/d (0.1%),167 mg/kg bw/d (0.25%)or 333 mg/kg bw/d (0.50%) over a period of 734 days.

Study 5: No data available

Details on mating procedure:

Study 2: - M/F ratio per cage:one male:one female
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy : The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. : N/A
- Further matings after two unsuccessful attempts: [no / yes (explain)] : no

Study 3: - M/F ratio per cage: 1:1
- Length of cohabitation: A maximum of 14 days
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy : The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation)
- Further matings after two unsuccessful attempts: [no / yes (explain)] : no
- After successful mating each pregnant female was caged (how): Mated females were housed individually during gestation and lactation.

Study 4: - M/F ratio per cage: 1:4
- Length of cohabitation: once a week for 3 weeks
- After successful mating each pregnant female was caged (how): Females were placed in individual cages.

Study 5: No data available

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Study 3: Gas Chromatography (GC) using an external standard technique.

Duration of treatment / exposure:

Study 2: 14 days prior to mating, throughout mating and gestation and continuing through lactation day 3.

Study 3: 48 consecutive days

Study 4: 734 days

Study 5: Male: 42 days
Female: 41 - 55 days (from 14 days before mating to day 4 of lactation)

Frequency of treatment:

Study 2: daily

Study 3: Daily

Study 4: Daily

Study 5: daily

Details on study schedule:

Study 2: No Data Available

Study 3: No Data Available

Study 4: No Data Available

Study 5: No Data Available

Remarks:

Study 2: Doses / Concentrations:
0, 50, 250 and 600 mg/kg-bw/day
Basis:
no data

Study 3: Doses / Concentrations:
0, 50, 250 and 600 mg/kg/day
Basis:
analytical conc.

Study 4: Doses / Concentrations:
0.0 (vehicle), 67 mg/kg bw/d (0.1%),167 mg/kg bw/d (0.25%) and 333 mg/kg bw/d (0.50%)
Basis:

Study 5: Doses / Concentrations:
0, 25, 75 or 250 mg/kg/day
Basis:
no data

No. of animals per sex per dose:

Study 2: Number of Animals per Dose: 20
0 mg/kg/day (control) - 10 males and 10 females
50 mg/kg/day - 10 males and 10 females
250 mg/kg/day - 10 males and 10 females
600 mg/kg/day - 10 males and 10 females

Study 3: Control: 10 males and 10 females
50 mg/kg/day: 10 males and 10 females
250 mg/kg/day: 10 males and 10 females
600 mg/kg/day: 10 males and 10 females

Study 4: PART I
Control: 3 males and 12 females
67 mg/kg bw /d (0.1%) : 3 males and 12 females
167 mg/kg bw/d (0.25%) : 3 males and 12 females
333 mg/kg bw/d (0.50%) :3 males and 12 females


PART II
Control: 3 males and 12 females
67 mg/kg bw/d (0.1%) : 3 males and 12 females
167 mg/kg bw /d (0.25%) : 3 males and 12 females
333 mg/kg bw/d (0.50%) :3 males and 12 females

PART III
Control: 3 males and 12 females
67 mg/kg bw/d (0.1%) : 3 males and 12 females
167 mg/kg bw/d (0.25%) : 3 males and 12 females
333 mg/kg bw/d (0.50%) :3 males and 12 females

Study 5: No data available

Control animals:

yes, concurrent vehicle

Details on study design:

Study 2: No Data Available

Study 3: No Data Available

Study 4: No Data Available

Study 5: No Data Available

Positive control:

No Data Available

Parental animals: Observations and examinations:

Study 2: Pregnancy and parturition: The following was recorded for each female:
i) Date of mating
jj) Date and time of observed start of parturition
iii) Date and time of observed completion of parturition
iv) Duration of gestation

Study 3: DETAILED CLINICAL OBSERVATIONS: Yes
All animals were examined for overt signs of toxicity, ill-health and behavioral change immediately before and after dosing, and one and 5 hours after dosing during the working week.

BODY WEIGHT: Yes
Individual body weights were recorded on Day 1 (prior to the start of treatment) and then weekly for males until termination. Females were weighed weekly until mating was evident. Body weights were recorded on Days 0, 7. 14 and 20 post coitumand on Days 1 and 4 post-parium.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
During the maturation period, weekly food consumption was recorded for each cage of adults. Food consumption was recorded for the periods covering Days 1-7, 7-14 and 14-21. For females with live litters, food consumption was recorded on Days 1 and 4 post-partum.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Water intake was observed daily by visual inspection of water bottles for any overt change.

OTHER:
reproductive function: corpora lutea and inplantation sites were examined.

Reproductive Indites:
Mating Index, pregnancy index and fertility index were examined.

organ weight:
Adrenals, Brain, Epididymides, Heart, Kidneys,, Liver, Ovaries, Spleen, Testes and Thymus were weighted.

Haematological and blood chemical observations :
Hematological and blood chemical investigations were performed on 5 males and 5 females selected from each test and control group on Day 14 (day prior to pairing).Blood samples were obtained from the lateral tail vein. Animals were not fasted prior to sampling.

Pregnancy and Parturition:
Each pregnant female was observed in the morning, around noon and in the afternoon and around the period of expected parturition. At weekends and public holidays, observations were carried out at in the morning and around noon. For each female date of mating, date and time of observed start of parturition, date and time of observed completion of parturition and duration of gestation was recorded.

Study 4: Following parameters were examined:
-Body weight and food consumption
-Live pups per litter
-Pup body weights
-Pup survival
-Histopathology
-Urine examination

-Gross Pathology : The 2-year ingestion of the test chemical caused lesions only in the urinary tract, namely, cystic dilatation of renal tubules with interstitial inflammation. Glomeruli were never altered. Lower tubules were sometimes filled with a proteinaceous fluid, and at other times with a blood-derived pigment. These accumulations were in some cases found in the renal pelvis or bladder, accompanied by mild epithelial hyperplasia or squamous metaplasia. The break-point for the occurrence of cystic change lies in the region of the 0.1% dietary concentration; and that for chronic nephritis, between 0.1 and 0.5%. The incidence of tumors was due to the senility of the rats at autopsy, not to treatment.

Study 5: Clinical sign, Body weight, Food consumption, Hematology, clinical chemistry and FOB were examined

Oestrous cyclicity (parental animals):

Study 2: No Data Available
Study 3: No Data Available
Study 4: No Data Available
Study 5: Changes in the estrous cyclicity were observed.

Sperm parameters (parental animals):

Study 2: No Data Available
Study 3: No Data Available
Study 4: No Data Available
Study 5: No data available

Litter observations:

Study 2: Offspring Litter Size and Viability

Study 3: Number of offspring born, Number and sex of offspring alive recorded daily and reported on Day 1 and 4 post partum, Clinical condition, Individual offspring and litter weights and surface righting reflex on Day 1 and 4 were observed.

Study 4: -Live pups per litter
-Pup body weights
-Pup survival
-Histopathology

Study 5: Pup Viability, Sex Ratio, Birth Index, Body weight of pups were observed.

Postmortem examinations (parental animals):

Study 2: No Data Available

Study 3: GROSS NECROPSY:All adult animals , including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Study 4: - Gross Pathology : The 2-year ingestion of the test chemical caused lesions only in the urinary tract, namely, cystic dilatation of renal tubules with interstitial inflammation. Glomeruli were never altered. Lower tubules were sometimes filled with a proteinaceous fluid, and at other times with a blood-derived pigment. These accumulations were in some cases found in the renal pelvis or bladder, accompanied by mild epithelial hyperplasia or squamous metaplasia. The break-point for the occurrence of cystic change lies in the region of the 0.1% dietary concentration; and that for chronic nephritis, between 0.1 and 0.5%. The incidence of tumors was due to the senility of the rats at autopsy, not to treatment.

Study 5: Organ weight and histopatholgy were examined

Postmortem examinations (offspring):

Study 2: No Data Available
Study 3: GROSS NECROPSY: All offspring were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
Study 4: -Live pups per litter
-Pup body weights
-Pup survival
-Histopathology
Study 5: No Data Available

Statistics:

Study 2: No Data Available
Study 3: Methods used included Dunnetts test or a pair wise Students t-test or Kruskal-Wallis non parametric one way analysis of variance and Mann Whitney “U” test/Wilcoxon signed rank test.
Study 4: No Data Available
Stduy 5: No Data Available

Reproductive indices:

Study 2: No Data Available
Study 3: No Data Available
Study 4: No Data Available
Study 5: Implantation Index, Resorption Index, Gestational Index.

Offspring viability indices:

Study 2: No Data Available
Study 3: No Data Available
Study 4: No Data Available
Study 5: Pup viability index.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Study 3: In 600 mg/kg/day, Increased salivation, pink staining was evident 00 cage tray liners from the male, red coloured urine, red brown staining around the snout and physical injury to the tail tip were observed pilo-erection, tiptoe gait, hunched posture, and skin pallor by day 11 and decrease respiratory rat on Day 12 the dose group terminated on Day 13.
in 250 mg/kg/day,Increased salivation and Hunched posture 'and tiptoe gait were observed in male and female rats as comarped to control.

Study 5: No effect was observed on clinical sign of treated male and female rats as compared to control.

Dermal irritation (if dermal study):

not specified

Mortality:

mortality observed, treatment-related

Description (incidence):

Study 3: When treated with 600 mg/kg/day, one male killed on day 13, on efemale dead on day 15, two on day 16 and all animals are sacrificed on day 17 of study. In 150 mg/kg/day dose group, significant decease in survival were observed and this dose level was terminated on Day 31.

Study 4: Respiratory infections were responsible for the few mortalities. The animals that were visibly ill between that time and the 2-year period were sacrificed and were autopsied.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Study 3: Slight reduction body weight gain were observed in 250 and 600 mg/kg/day treated rats as compared to control. In 50 mg/kg/day, no statistical significance effect were observed in treated rats as compared to control.

Study 4: Mean weights of males were found to be deviated at 0.1% dose group while, no changes in weights were observed in females from all the dose groups.

Study 5: Body weight: No effect was observed on body weight of treated male and female rats as compared to control.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Study 3: Slight reduction body weight gain were observed in 250 and 600 mg/kg/day treated rats as compared to control. In 50 mg/kg/day, no statistical significance effect were observed in treated rats as compared to control.

Study 4: No effects were seen on food consumption

Study 5: Food consumption: No effect was observed on food consumption of treated male and female rats as compared to control.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Study 3: A significantly lower platelet count was apparent for males receiving 250 mg/kg/day compared to controls. For females treated with 250 mg/kg/day, platelet count at pre-mating assessment was only marginally lower than controls.
Males receiving 50 or 250 mg/kg/day showed a significant reduction in white blood cell count at pre-mating assessment, compared with control. No similar effects were observed for females.

Study 5: In male rats, when treated with 75 mg/kg/day, prolongation in the PT and APTT were observed as compared to control. When treated with 250 mg/kg/day, prolongation in APTT were observed as compared to control. In female rats, decrease in the RBC and MCHC and increase in the RET was observed at recovery(250 mg/kg/day) as compared to control

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Study 5: No effect was observed on clinical chemistry of treated male and female rats as compared to control.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Study 4: At 333 mg/kg bw/d (0.50%) :Prolonged test chemical ingestion did not increase protein excretion by rats fed as much as 0.50%

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Study 5: No effect was observed on functional observation battery (FOB) of treated male and female rats as compared to control.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Study 3: When treated with 600 mg/kg/day, Higher grades of severity of trabecular bone formation in female , Centrilobular hepatocyte enlargement of liver, Hypertrophy of the collecting duct epithelium of the renal papilla and extramadullary haemopiesis of spleen were observed male and female rats. When treated with 250 mg/kg/day, Higher grades of severity of trabecular bone formation in female , Centrilobular hepatocyte enlargement of liver in male and female, Hypertrophy of the collecting duct epithelium of the renal papilla and extramadullary haemopiesis of spleen were observed female rats as compared to control.

Study 5: No Histopathological changes were observed in treated male and female rats as compared to control.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Study 5: No changes were observed in the estrous cycles of female rats.

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

Study 2: No adverse effects on mating performance, fertility or gestation were detected.
Study 3: No effect were observed on mating performance or fertility and corpora lutea and implantation of treated rats as compared to control.
Study 4: no effects observed
Study 5: no effects observed

Study 2: No adverse effects on mating performance, fertility or gestation were detected.

Study 3: Clinical toxicity:
mortality:
when treated with 600 mg/kg/day, one male killed on day 13, on efemale dead on day 15, two on day 16 and all animals are sacrificed on day 17 of study. In 150 ma/kg/day dose group, significant decease in survival were observed and this dose level was terminated on Day 31

Clinical signs:
in 600 mg/kg/day, Increased salivation, pink staining was evident 00 cage tray liners from the male, red coloured urine, red brown staining around the snout and physical injury to the tail tip were observed pilo-erection, tiptoe gait, hunched posture, and skin pallor by day 11 and decrease respiratory rat on Day 12 the dose group terminated on Day 13.
in 250 mg/kg/day,Increased salivation and Hunched posture 'and tiptoe gait were observed in male and female rats as comarped to control.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No adverse effect was detected for males throughout the treatment period or for females during maturation or gestation. However, a slight reduction in body weight gain was observed for females treated with 125 mg/kg/day during lactation.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
No adverse effect was detected for males. There were no adverse effects on dietary intake or food efficiency for females throughout the maturation or gestation phases of the study. However, during lactation reduced dietary intake was detected for females receiving 125 mg/kg/day compared with controls.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There was no effect on mating or fertility, however, a shorter gestation length was observed in females treated with 25 and 125 mg/kg/day.

OTHER FINDINGS (PARENTAL ANIMALS) :

Haematology
A significantly lower platelet count was apparent for males receiving 250 mg/kg/day compared to controls. For females treated with 250 mg/kg/day, platelet count at pre-mating assessment was only marginally lower than controls.
Males receiving 50 or 250 mg/kg/day showed a significant reduction in white blood cell count at pre-mating assessment, compared with control. No similar effects were observed for females.

Blood Chemistry
Clear reductions in total protein, albumin. and albumin:globulin ratio, compared to control levels were apparent for both sexes at 250 mg/kg/day, with similar changes also being observed, to a lesser degree, for both sexes at 50 mg/kg/day. Alkaline phosphatase levels during pre-mating and terminal assessments were elevated, in comparison to control for both sexes at 250 rug/kg/day and for males at 5 mg/kg/day.

Gross Pathalogy:
In 600 mg/kg/day dose group, skin pallor, pillo-erection, and staining around the snout, and pallor of lings, spleen, stomach, liver, small intestine and dark enlarge liver, thymus, kidney, left testis and prostate of treated male and female rats.
in 250 mg/kg/day dose group, pallor of the extremities and vaginal bleeding and all tissues was pale and dark contents evident in gastro intestinal and uterus with red fluid. Liver pallor was prevalent for animals of either sex treated at this dose level.
no effect were observed in 50 mg/kg/day dose group as comapred to control.

Histopathology:
When treated with 600 mg/kg/day, Higher grades of severity of trabecular bone formation in female , Centrilobular hepatocyte enlargement of liver, Hypertrophy of the collecting duct epithelium of the renal papilla and extramadullary haemopiesis of spleen were observed male and female rats.
when treated with 250 mg/kg/day, Higher grades of severity of trabecular bone formation in female , Centrilobular hepatocyte enlargement of liver in male and female, Hypertrophy of the collecting duct epithelium of the renal papilla and extramadullary haemopiesis of spleen were observed female rats as compared to control.

Study 4: FOOD CONSUMPTION (PARENTAL ANIMALS):-No effects were seen on food consumption
GROSS PATHOLOGY (PARENTAL ANIMALS) :-At 67 mg/kg bw/d (0.1%) and 333 mg/kg bw/d (0.50%) :A lesion characteristically seen in the kidneys of untreated old rats becomes greatly exaggerated in degree.
At dietary levels of 0.01% and below : the renal lesions present were indistinguishable from those of the control group.
URINE EXAMINATION:At 333 mg/kg bw/d (0.50%) :Prolonged test chemical ingestion did not increase protein excretion by rats fed as much as 0.50%
EFFECT ON GROWTH: At 333 mg/kg bw/d (0.50%) - caused growth arrests which were partly due to lowered food intakes.
At 67 mg/kg bw/d (0.1%) :Caused no growth inhibition in either sex.
At 333 mg/kg bw/d (0.50%) : Distinct growth inhibition observed.

Study 5: Haematology:
In male rats, when treated with 75 mg/kg/day, prolongation in the PT and APTT were observed as compared to control.
When treated with 250 mg/kg/day, prolongation in APTT were observed as compared to control.
In female rats, decrease in the RBC and MCHC and increase in the RET was observed at recovery (250 mg/kg/day) as compared to control
Clinical chemistry
No effect was observed on clinical chemistry of treated male and female rats as compared to control.
Neurobehaviour
No effect was observed on functional observation battery (FOB) of treated male and female rats as compared to control.

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects on mating performance, fertility or gestation were observed.

Remarks on result:

other: Not Specified

Critical effects observed:

not specified

System:

other: Not Specified

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Study 2: No effect were observed on growth of 50 mg/kg/day treated offspring as compared to control.

Dermal irritation (if dermal study):

not specified

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Study 2: Females treated with 600 mg/kg/day showed a higher percentage of pre-implantation losses in comparison to controls, resulting in the birth of less offspring per litter and lower total litter weights at this dose level.

Study 3: The number of interim offspring deaths at 50 mg/kg/day was considerably higher than observed for controls.

Study 4: No test chemical related mortality was observed.

Study 5: no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Study 3: Offspring weights on Day 1 and subsequent weight gain at lower dose levels were similar to control and unaffected by treatment. For offspring at 250 mg/kg/day, bodyweight on Day 1 of age was slightly lower than control; differences failed to attain statistical significance and may reflect the generally shorter gestation length for litters at this dose level.

Study 4: Decreased weights of the pups was observed probably due to inadequate food intakes by their gestating and lactating mothers fed with test chemical through diet.

Study 5: The test chemical had no effects on the body weight of the pups.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Study 3: Males treated with 250 mg/kg/day showed elevated liver weights in comparison to controls, however, statistical significance was not achieved. Females treated with 250 mg/kg/day showed a statistically significant reduction in spleen weights.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Study 3: Necropsy
Interim death and terminal kill offspring did not show any abnormalities considered to be related to treatment. Two males treated with 250 mg/kg/day displayed liver pallor at terminal kill.

Study 5: no effects observed

Histopathological findings:

not specified

Description (incidence and severity):

Study 3: Minimal centrilobular hepatocyte enlargement 'was observed in relation to treatment for animals of either sex treated with 250 mg/kg/day. Females treated with 50 mg/kg/day were similarly affected.

Study 5: no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Study 4: Litter observations :-
-There was no effect on the number of litters born nor on mortality of the offspring.
-The average size of the litters decreased as the concentration of dietary intake of the test chemical increased,with the anomalous exception of the numbers of pups born and weaned in the second generation whose mothers were fed the 67 mg/kg bw/d (0.1%) diet.
-At 333 mg/kg bw/d (0.50%) :
Reduced litter size and the weight of the youngs at 21 days was observed.
Survival:-
At 67 mg/kg bw/d (0.1%) and 333 mg/kg bw/d (0.50%) :There was good survival in all groups, with no significant reduction in the number of survivors at any level of the test chemical.

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

Study 2: Offspring Litter Size and Viability: Females treated with 600 mg/kg/day showed a higher percentage of pre-implantation losses in comparison to controls, resulting in the birth of less offspring per litter and lower total litter weights at this dose level.

Study 3: BODY WEIGHT (OFFSPRING):
Offspring weights on Day 1 and subsequent weight gain at lower dose levels were similar to control and unaffected by treatment. For offspring at 125 mg/kg/day, bodyweight on Day 1 of age was slightly lower than control; differences failed to attain statistical significance and may reflect the generally shorter gestation length for litters at this dose level.
ORGAN WEIGHTS (OFFSPRING)
Males treated with 125 mg/kg/day showed elevated liver weights in comparison to controls, however, statistical significance was not achieved. Females treated with 125 mg/kg/day showed a statistically significant reduction in spleen weights.
GROSS PATHOLOGY (OFFSPRING)
Necropsy
Interim death and terminal kill offspring did not show any abnormalities considered to be related to treatment. Two males treated with 125 mg/kg/day displayed liver pallor at terminal kill.
HISTOPATHOLOGY (OFFSPRING)
Minimal centrilobular hepatocyte enlargement 'was observed in relation to treatment for animals of either sex treated with 125 mg/kg/day. Females treated with 25 mg/kg/day were similarly affected.
OTHER FINDINGS (OFFSPRING)
Mortality :The number of interim offspring deaths at 25 and 125 mg/kg/day was considerably higher than observed for controls.

Study 4: Body weight :- At weaning: Decreased weights of the pups was observed probably due to inadequate food intakes by their gestating and lactating mothers with dietary intake of the test chemical.
Litter observations :-
-There was no effect on the number of litters born nor on mortality of the offspring.
-The average size of the litters decreased as the concentration of dietary intake of the test chemical increased,with the anomalous exception of the numbers of pups born and weaned in the second generation whose mothers were fed the 67 mg/kg bw/d (0.1%) diet.
-At 333 mg/kg bw/d (0.50%) :
Reduced litter size and the weight of the youngs at 21 days was observed.
Survival:-
At 67 mg/kg bw/d (0.1%) and 333 mg/kg bw/d (0.50%) :There was good survival in all groups, with no significant reduction in the number of survivors at any level of the test chemical.

Study 5: No effect of the test chemical was observed on live pup index, viability index, and external anomalies.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

mortality

Remarks on result:

other: Not Specified

Critical effects observed:

not specified

System:

other: Not Specified

Reproductive effects observed:

not specified

Treatment related:

not specified

Conclusions:

Study 2: Based on all the observation and results, it was concluded that the NOAEL of the test chemical was 250 mg/kg bw after administration to the test animals.

Study 3: NOAEL was considered to be 50 mg/kg/day for male and LOAEL for female F0 generation and NOAEL F1 ganaration when Sprague-Dawley Crl: CD (SD) IGS BR male and female rats treated with the test chemical.

Study 4: Based on all the observations and results, it was concluded that the NOAEL for the test chemical is found to be 67 mg/kg bw (0.1%).

Study 5: Based on all the observations and results, it was concluded that the NOAEL was considered to be 250 mg/kg/day when Crl:CD (SD) male and female rat were treated with the test chemical.

Executive summary:

Reproductive Toxicity Study:

The summaries of the reproductive toxicity data is as follows:

Reproductive Toxicity Study 2:

In the combined repeated-dose/reproductive/developmental toxicity screening study, the test material was administered by gavage to three groups each of ten male and ten female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for up to fifty-four consecutive days, at dose levels of 50, 250 and 600 mg/kg/day. The following was recorded for each female: Date of mating, Date and time of observed start of parturition, Date and time of observed completion of parturitionand Duration of gestation. After dosing, it was observed that,Females treated with 600 mg/kg/day showed a higher percentage of pre-implantation losses in comparison to controls, resulting in the birth of less offspring per litter and lower total litter weights at this dose level. Thus, no adverse effects (NOAEL) on mating performance, fertility or gestation were observed at 250 mg/kg-bw/day of concentartion. Also, the same NOAEL was considered for fetal parameters. Thus, based on all the observation and results, it was concluded that the NOAEL of the test chemical was 250 mg/kg bw after administration to the test animals.

Reproductive Toxicity Study 3:

In a combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test, Sprague-DawleyCrl: CD (SD) IGS BR male and female rats treated with the test chemical in the concentration of 0, 50, 250 and 500 mg/kg/day orally by gavage. one male killed on day 13, on efemale dead on day 15, two on day 16 and all animals are sacrificed on day 17 of study at 600 mg/kg/day dose group and significant decease in survival were observed and this dose level was terminated on Day 31 at 250 ma/kg/day dose group. Increased salivation, pink staining was evident 00 cage tray liners from the male, red coloured urine, red brown staining around the snout and physical injury to the tail tip were observed. pilo-erection, tiptoe gait, hunched posture, and skin pallor by day 11 and decrease respiratory rat on Day 12 the dose group terminated on Day 13 in 600 mg/kg/day and Increased salivation and Hunched posture 'and tiptoe gait were observed in male and female rats as compared to control in 250 mg/kg/day dose group. slight reduction body weight gain were observed in 250 and 600 mg/kg/day treated rats as compared to control. in addition, skin pallor, pillo-erection, and staining around the snout, and pallor of lings, spleen, stomach, liver, small intestine and dark enlarge liver, thymus, kidney, left testis and prostate of treated male and female rats at 600 mg/kg/day and pallor of the extremities and vaginal bleeding and all tissues was pale and dark contents evident in gastro intestinal and uterus with red fluid. Liver pallor was prevalent for animals of either sex treated at this dose level at 250 mg/kg/day dose group. Higher grades of severity of trabecular bone formation in female , Centrilobular hepatocyte enlargement of liver, Hypertrophy of the collecting duct epithelium of the renal papilla and extramadullary haemopiesis of spleen in 600 mg/kg/day dose group and Higher grades of severity of trabecular bone formation in female , Centrilobular hepatocyte enlargement of liver in male and female, Hypertrophy of the collecting duct epithelium of the renal papilla and extramadullary haemopiesis of spleen were observed female rats as compared to control at 250 mg/kg/day dose group. no effect were observed on mating performance or fertility and corpora lutea and implantation of 50 mg/kg/day treated rats as compared to control. no effect were observed on offspring Viability, clinical sign and body weight of offsprings as compared to control. therefore NOAEL was considered to be 50 mg/kg/day for male and NOAEL for female F0 generation and NOAEL F1 ganaration when Sprague-DawleyCrl: CD (SD) IGS BR male and female rats were treated with the test chemical orally by gavage.

Reproductive Toxicity Study 4:

Two-year feeding of 0.5 and 1.0% dietary levels of the test chemical to male and female albino rats caused growth arrests which were partly due to lowered food intakes. The females’ growth was slightly arrested at 0.1%, an effect apparently due to the test chemical itself. Any such effect at 0.1% on the males could have been obscured by the greater deviations of their weights from their mean weights than the deviations of the females’ weights. Moderate degrees of anemia resulted from prolonged ingestion of 0.5 and 1.0% concentrations of the test chemical, but prompt recoveries of hemoglobin followed feeding of the control diet. Leukocyte numbers and percentages remained within normal ranges. The test chemical did not increase proteinuria or cause glycosuria. It is highly probable that apparent decreases in litter sizes and numbers of pups weaned were largely due to inadequate food intakes by their gestating and lactating mothers fed with the test chemical. The significantly decreased weights of the pups, at weaning, in but one of the nine experimental dietary categories was probably due to the same cause. The 2-year ingestion of the test chemical caused lesions only in the urinary tract, namely, cystic dilatation of renal tubules with interstitial inflammation. Glomeruli were never altered. Lower tubules were sometimes filled with a proteinaceous fluid, and at other times with a blood-derived pigment. These accumulations were in some cases found in the renal pelvis or bladder, accompanied by mild epithelial hyperplasia or squamous metaplasia. The break-point for the occurrence of cystic change lies in the region of the 0.1% dietary concentration; and that for chronic nephritis, between 0.1 and 0.5%. Thus, based on all the observations and results, it was concluded that the NOAEL for the test chemical is found to be 67 mg/kg bw (0.1%).

Reproductive Toxicity Study 5:

In a combined repeated dose and reproduction/developmental screening test, male and female Crl:CD (SD) rats were treated orally with the test chemical in concentration of 0, 25, 75 or 250 mg/kg/day with recovery dose group of 0 and 250 mg/kg/day.No effect were observe on clinical sign, body weight and food consumption of treated rats as compared to control. Similarly, no effects were observed on blood chemistry, organ weight and histopathology of treated rats as compared to control. In addition, changes were observed as tendency to prolongation in the PT and prolongation in the APTT in male rats at 75 and 250 mg/kg/day dose group and decrease in the RBC and MCHC. An increase in the RET at 250 mg/kg/day dose group during recovery in female rats as compared to control. No reproductive effect were observed in treated male or female rats and on offspring’s as compared to control. Therefore,NOAEL for the parental generation and the F1 generation were considered to be 250 mg/kg/day when male and female Crl:CD (SD) rats were treated orally by gavage with the test chemical.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The data is from a Klimisch 2 database and provides a robust study summary.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Reproductive Toxicity Study:

The summaries of the reproductive toxicity data is as follows:

Reproductive Toxicity Study 2:

In the combined repeated-dose/reproductive/developmental toxicity screening study, the test material was administered by gavage to three groups each of ten male and ten female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for up to fifty-four consecutive days, at dose levels of 50, 250 and 600 mg/kg/day. The following was recorded for each female: Date of mating, Date and time of observed start of parturition, Date and time of observed completion of parturitionand Duration of gestation. After dosing, it was observed that,Females treated with 600 mg/kg/day showed a higher percentage of pre-implantation losses in comparison to controls, resulting in the birth of less offspring per litter and lower total litter weights at this dose level. Thus, no adverse effects (NOAEL) on mating performance, fertility or gestation were observed at 250 mg/kg-bw/day of concentartion. Also, the same NOAEL was considered for fetal parameters. Thus, based on all the observation and results, it was concluded that the NOAEL of the test chemical was 250 mg/kg bw after administration to the test animals.

Reproductive Toxicity Study 3:

In a combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test, Sprague-DawleyCrl: CD (SD) IGS BR male and female rats treated with the test chemical in the concentration of 0, 50, 250 and 500 mg/kg/day orally by gavage. one male killed on day 13, on efemale dead on day 15, two on day 16 and all animals are sacrificed on day 17 of study at 600 mg/kg/day dose group and significant decease in survival were observed and this dose level was terminated on Day 31 at 250 ma/kg/day dose group. Increased salivation, pink staining was evident 00 cage tray liners from the male, red coloured urine, red brown staining around the snout and physical injury to the tail tip were observed. pilo-erection, tiptoe gait, hunched posture, and skin pallor by day 11 and decrease respiratory rat on Day 12 the dose group terminated on Day 13 in 600 mg/kg/day and Increased salivation and Hunched posture 'and tiptoe gait were observed in male and female rats as compared to control in 250 mg/kg/day dose group. slight reduction body weight gain were observed in 250 and 600 mg/kg/day treated rats as compared to control. in addition, skin pallor, pillo-erection, and staining around the snout, and pallor of lings, spleen, stomach, liver, small intestine and dark enlarge liver, thymus, kidney, left testis and prostate of treated male and female rats at 600 mg/kg/day and pallor of the extremities and vaginal bleeding and all tissues was pale and dark contents evident in gastro intestinal and uterus with red fluid. Liver pallor was prevalent for animals of either sex treated at this dose level at 250 mg/kg/day dose group. Higher grades of severity of trabecular bone formation in female , Centrilobular hepatocyte enlargement of liver, Hypertrophy of the collecting duct epithelium of the renal papilla and extramadullary haemopiesis of spleen in 600 mg/kg/day dose group and Higher grades of severity of trabecular bone formation in female , Centrilobular hepatocyte enlargement of liver in male and female, Hypertrophy of the collecting duct epithelium of the renal papilla and extramadullary haemopiesis of spleen were observed female rats as compared to control at 250 mg/kg/day dose group. no effect were observed on mating performance or fertility and corpora lutea and implantation of 50 mg/kg/day treated rats as compared to control. no effect were observed on offspring Viability, clinical sign and body weight of offsprings as compared to control. therefore NOAEL was considered to be 50 mg/kg/day for male and NOAEL for female F0 generation and NOAEL F1 ganaration when Sprague-DawleyCrl: CD (SD) IGS BR male and female rats were treated with the test chemical orally by gavage.

Reproductive Toxicity Study 4:

Two-year feeding of 0.5 and 1.0% dietary levels of the test chemical to male and female albino rats caused growth arrests which were partly due to lowered food intakes. The females’ growth was slightly arrested at 0.1%, an effect apparently due to the test chemical itself. Any such effect at 0.1% on the males could have been obscured by the greater deviations of their weights from their mean weights than the deviations of the females’ weights. Moderate degrees of anemia resulted from prolonged ingestion of 0.5 and 1.0% concentrations of the test chemical, but prompt recoveries of hemoglobin followed feeding of the control diet. Leukocyte numbers and percentages remained within normal ranges. The test chemical did not increase proteinuria or cause glycosuria. It is highly probable that apparent decreases in litter sizes and numbers of pups weaned were largely due to inadequate food intakes by their gestating and lactating mothers fed with the test chemical. The significantly decreased weights of the pups, at weaning, in but one of the nine experimental dietary categories was probably due to the same cause. The 2-year ingestion of the test chemical caused lesions only in the urinary tract, namely, cystic dilatation of renal tubules with interstitial inflammation. Glomeruli were never altered. Lower tubules were sometimes filled with a proteinaceous fluid, and at other times with a blood-derived pigment. These accumulations were in some cases found in the renal pelvis or bladder, accompanied by mild epithelial hyperplasia or squamous metaplasia. The break-point for the occurrence of cystic change lies in the region of the 0.1% dietary concentration; and that for chronic nephritis, between 0.1 and 0.5%. Thus, based on all the observations and results, it was concluded that the NOAEL for the test chemical is found to be 67 mg/kg bw (0.1%).

Reproductive Toxicity Study 5:

In a combined repeated dose and reproduction/developmental screening test, male and female Crl:CD (SD) rats were treated orally with the test chemical in concentration of 0, 25, 75 or 250 mg/kg/day with recovery dose group of 0 and 250 mg/kg/day.No effect were observe on clinical sign, body weight and food consumption of treated rats as compared to control. Similarly, no effects were observed on blood chemistry, organ weight and histopathology of treated rats as compared to control. In addition, changes were observed as tendency to prolongation in the PT and prolongation in the APTT in male rats at 75 and 250 mg/kg/day dose group and decrease in the RBC and MCHC. An increase in the RET at 250 mg/kg/day dose group during recovery in female rats as compared to control. No reproductive effect were observed in treated male or female rats and on offspring’s as compared to control. Therefore,NOAEL for the parental generation and the F1 generation were considered to be 250 mg/kg/day when male and female Crl:CD (SD) rats were treated orally by gavage with the test chemical.

Effects on developmental toxicity

Description of key information

Developmental Toxicity Study:

In the combined repeated-dose/reproductive/developmental toxicity screening study, the test material was administered by gavage to three groups each of ten male and ten female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for up to fifty-four consecutive days, at dose levels of 50, 250 and 600 mg/kg/day. The following was recorded for each female: Date of mating, Date and time of observed start of parturition, Date and time of observed completion of parturitionand Duration of gestation. After dosing, it was observed that,Females treated with 600 mg/kg/day showed a higher percentage of pre-implantation losses in comparison to controls, resulting in the birth of less offspring per litter and lower total litter weights at this dose level. Thus, no adverse effects (NOAEL) on mating performance, fertility or gestation were observed at 250 mg/kg-bw/day of concentartion. Also, the same NOAEL was considered for fetal parameters. Thus, based on all the observation and results, it was concluded that the NOAEL of the test chemical for the maternal and fetal parameters considered to be 250 mg/kg bw after administration to the test animals.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Weight of evidence based on the information about the test chemical.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Principles of method if other than guideline:

Equivalent or similar to OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Species:

other: Study 2: rat ; Study 3: rabbit Study 4: rat

Strain:

other: Study 2: Sprague-Dawley ; Study 3: New Zealand White Study 4: Crj: CD(SD)

Details on test animals or test system and environmental conditions:

Study 2: No Data Available

Study 3: TEST ANIMALS
- Source:Cheshire Rabbit Farms (Oudden Lodge. Nr. Tarporley.Cheshire). Ranch Rabbits (Crawley Down.Sussex). and Buxted Rabbit Co. Ltd (Gt. Totease Farm, Buxted. Sussex).
- Age at study initiation for p: Twelve to twenty weeks of age
- Weight at study initiation:2.9 to 4.1 kg
- Housing:The rabbits were housed individually in metal cages equipped with sheet stainless steel sides, back and top, a stainless steel wire front and an aluminium perforated floor panel.Individual undercage plastic trays were lined with absorbent paper which was inspected and changed daily.
- Diet (e.g. ad libitum):SDS Rabbit Diet SQC,ad libitum
- Water (e.g. ad libitum):Tap water,ad libitum
- Acclimation period:10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C):18°C ± 3°C
- Humidity (%): 55% ± 15%
- Photoperiod (hrs dark / hrs light): 14 hours


Study 4: Details on test animal
TEST ANIMALS
- Source: No data available
- Age at study initiation: (P) x wks; (F1) x wks: P- 9 weeks old
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g: No data available

- Fasting period before study: No data available
- Housing: No data available
- Diet (e.g. ad libitum): No data available
- Water (e.g. ad libitum): No data available
- Acclimation period: No data available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data available
- Humidity (%):No data available
- Air changes (per hr): No data available
- Photoperiod (hrs dark / hrs light): No data available

Route of administration:

other: Study 2: oral: gavage; Study 3: oral: gavage; Study 4: oral: gavage

Vehicle:

other: Study 2: corn oil; Study 3: 1% Methyl-cellulose; Study 4: Not Specified

Details on exposure:

Study 2: The test material was administered by gavage to three groups each of ten male and ten female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for up to fifty-four consecutive days, at dose levels of 50, 250 and 600 mg/kg/day.

Study 3: Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The dosages of diphenylamine administered by oral gavage in the present study were 33,100 and 300 mg/kg bodyweight/day. Treatment by intragastric intubation commenced on Day 7 of pregnancy and continued daily up to and including Day 19 of gestation. The highest required concentration of suspension was prepared by suspending the test material in the vehicle. The lower concentrations were prepared by serial dilution with the vehicle. The suspensions were prepared freshly each day. Treatment by intragastric intubation commenced on Day 7 of pregnancy and continued daily up to and including Day 19 of gestation. Dose volumes were calculated for individual animals on Day 7 of pregnancy and adjusted according to bodyweight on Days 9, 11 and 15.
VEHICLE : Methyl-cellulose

Study 4: No data available

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

Study 2: No Data Available

Study 3: No Data Available

Study 4: No Data Available

Details on mating procedure:

Study 2:
- M/F ratio per cage: 1:1
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy : The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. : N/A
- Further matings after two unsuccessful attempts: [no / yes (explain)] : no

Study 3:
Details of mating : Does were mated with males of proven fertility: once coitus had been observed, each female was allowed to remain with the male for at least one hour. Sixty-six of those dams which successfully completed coituswere each injected intravenously with 25 i.u. of Chorulon (luteinizing hormone) to promote ovulation. The day of mating was considered as Day 0 of pregnancy.

- Mating was phased over consecutive week days (Monday to Thursday) ensuring that no more than 20 animals were mated on anyone day. On each of these days, mated does were allocated to four groups equalising distribution as far as possible, as to number allocated,source of animals and male to which they were mated.

- Length of cohabitation:1 hour

- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: The day of mating was considered as Day 0 of pregnancy.

- Further matings after two unsuccessful attempts: [no / yes (explain)] : No

- After successful mating each pregnant female was caged (how): Females were re-assigned to cages in the experimental room.

- Any other deviations from standard protocol: Structural deviations were classified as:
Malformations : rare and/or probably lethal, e.g. amelia,exencephaly.
Anomalies : minor differences from 'normal' that are detected relatively frequently either at initial examination. e.g. variations of the gall bladder, or at skeletal examination. e.g. hemicentric vertebra.

-Variants : alternative structures occurring regularly in the control population are classified as variants. These may be permanent structures e.g. an extra pair of ribs, or they may be transient stages of development. e.g. unossified sternebra(e).

Study 4: No Data Available

Duration of treatment / exposure:

Study 2:
Males: 43 days;
Females: up to 54 days

Study 3: Days 7 to 19 of gestation

Study 4: Male: 42 days
Female: 41 - 55 days (from 14 days before mating to day 4 of lactation)

Frequency of treatment:

Study 2: Daily
Study 3: daily
Study 4: daily

Duration of test:

Study 2: 54 days prior to mating, throughout mating and gestation and continuing through lactation day 3
Study 3: 29 days
Study 4: No Data Available

Remarks:

Study 2: Doses / Concentrations:
0, 50, 250, and 600 mg/kg/day
Basis:

Study 3: Doses / Concentrations:
0, 33, 100 and 300 mg/kg/day
Basis:

Study 4: Doses / Concentrations:
0, 25, 75 or 250 mg/kg/day
Basis:
no data

No. of animals per sex per dose:

Study 2: 20 animals in each group
0 mg/kg/day (control) - ten male and ten female Sprague-Dawley rats
50 mg/kg/day - ten male and ten female Sprague-Dawley rats
250 mg/kg/day - ten male and ten female Sprague-Dawley rats
600 mg/kg/day - ten male and ten female Sprague-Dawley rats

Study 3: Control - 16 females
33 mg/kg/day - 16 females
100 mg/kg/day – 18 females
300 mg/kg/day – 16 females

Study 4: No data available

Control animals:

yes, concurrent vehicle

Details on study design:

Study 2: No Data Available

Study 3: No Data Available

Study 4: No Data Available

Maternal examinations:

Study 2: Survival, clinical sign, body weight, food consumption, water consumption, Duration of gestation, corpora luteaand, implantation site, fatality, hematology, clinical chemistry, oragn weight, gross patholology, histopathology and reperoductive performance were observed.
Pregnancy and parturition: The following was recorded for each female:
i) Date of mating
jj) Date and time of observed start of parturition
iii) Date and time of observed completion of parturition
iv) Duration of gestation

Study 3: Maternal examinations
a) Clinical observations: All animals were regularly handled and observed daily;obvious changes or signs of reaction to treatment were recorded.

b) Mortalities : Any animals that died or were killed for reasons of animal welfare were weighed and subjected to post mortem examination.

At 100 and 300 mg/kg bw dose levels : No increase in mortality

c) Body weight: All rabbits were weighed on Days 1, 7, 9, 11, 15, 20, 24 and 29 of gestation.
At 300 mg/kg bw/day : Reduction of mean body weight

d) Food consumption: The food intake of all rabbits was measured from weigh-day to weigh-day.

At 300 mg/kg bw/day : decrease in mean food consumption observed.

Post-mortem examinations: Yes
- Sacrifice on gestation day 29
- Organs examined: ovaries and uteri
the foetuses were removed by caesarean section and examined for visceral and skeletal anomalies.

Study 4: Clinical sign, Body weight, Food consumption, Hematology, clinical chemistry and FOB were examined

Ovaries and uterine content:

Study 2: Duration of gestation, corpora lutea and, implantation site was observed.

Study 3:
The uterine content was examined after termination: Yes
Examinations included:
number and distribution of live young: Yes
Gravid uterus weight: No
Number of corpora lutea: Yes
Number of implantations: Yes

Study 4: Changes in the estros cyclicity were observed.

Fetal examinations:

Study 2: Survival, clinical sign, body weight and development were observed.

Study 3:
Fetal examinations
Individual foetal weight: Yes
Sex: Yes
Number and distribution of embryonic/foetal deaths: Yes
Embryonic/foetal deaths were classified as:
Early: only placenta visible at termination
Late: both placenta and embryonic remnants visible at
termination
Foetal abnormalities : Yes
Visceral and skeletal anomalies : Yes

Study 4: No data available

Statistics:

Study 2: No Data Available

Study 3: Statistical analyses were performed routinely on litter data and incidences of skeletal anomalies and skeletal variants using the litter as the basic sample unit. Non-parametric methods (Kruskal-Wallis and Jonckheere tests) , were employed since these values rarely follow a normal distribution.

Study 4: No data available

Indices:

Study 2: Implantation Index, Gestational Index, Pup viability/survival index.

Study 3: Group mean values for litter size. embryonic death. pre- and post implantation loss were calculated in two ways:
Mean A: includes all surviving animals that provide evidence of pregnancy including those showing abortion or total
resorption.
Mean B: includes all animals with live young at termination.
Mean B has more meaning when group size is low in which case the mean values would be unduly influenced by the presence of a single animal with total litter loss.
Mean A is a more accurate index when several animals show total litter loss.
For each litter:
Pre-implantation loss was calculated as a percentage, from the formula:
(No. of corpora lutea - No. of implantations)/No. of corpora Iutea x 100
Post implantation loss was similarly calculated from the formula:
(No. of implantations - No. of live young)/NO. of implantations x 100
For each group:
All values expressed as a percentage or ratio were first
calculated within each litter and the group values derived as a mean of individual litter percentages.

For sex ratios of pups, litter weights, mean foetal weights and abnormality values, only Mean B were calculated.

Study 4: Implantation Index, Resorption Index, Gestational Index, Pup viability index.

Historical control data:

No Data Available

Clinical signs:

no effects observed

Description (incidence and severity):

Study 2: no effects observed

Study 3: At all dosages but in particular at 100 and 300 mg/kg/day, treatment was generally associated with green discolouration of the urine. There were no other signs of reaction considered attributable to treatment.

Study 4: No effect was observed on clinical sign of treated male and female rats as compared to control.

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

Study 2: no mortality observed

Study 3: Occasional animals were killed, found dead or excluded from the study for reasons of animal welfare. No association with treatment was indicated.

Study 4: No data Available

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Study 3: At 300 mg/kg/day animals showed a slight initial mean weight loss from the start of treatment. Subsequently mean weight gain remained slightly lower than among control animals. Differences in mean weight gains at 33 and 100 mg/kg/day were not dosage related; overall weight gain was lower than for control animals at 33 mg/kg/day but was slightly higher at 100 mg/kg/day.

Study 4: Body weight: No effect was observed on body weight of treated male and female rats as compared to control.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Study 2: At 300 mg/kg/day mean food consumption was reduced relative to control values. Differences from control values at 33 and 100 mg/kg/day showed no apparent relationship to dosage.

Study 4: Food consumption: No effect was observed on food consumption of treated male and female rats as compared to control.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Study 4: In female rats, decrease in the RBC and MCHC and increase in the RET was observed at recovery(250 mg/kg/day) as compared to control

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Study 4: No effect was observed on clinical chemistry of treated male and female rats as compared to control.

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Study 4: No effect was observed on functional observation battery (FOB) of treated male and female rats as compared to control.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Study 4: No effect was observed on body weight of treated male and female rats as compared to control.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Study 3: no effects observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Study 3: no effects observed
Study 4: No Histopathological changes were observed in treated male and female rats as compared to control.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

Study 3: No significant effects were observed by the administration of the test chemical in the animals.

Number of abortions:

no effects observed

Description (incidence and severity):

Study 2: no effects observed
Study 4: no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Study 2: no effects observed
Study 4: no effects observed

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Description (incidence and severity):

Study 2: no effects observed
Study 4: no effects observed

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Study 3: no effects observed
Study 4: no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

Study 2: no effects observed
Study 3: no effects observed
Study 4: no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Study 2: no effects observed
Study 4: no effects observed

Details on maternal toxic effects:

Study 2: Maternal toxic effects:no effects
Details on maternal toxic effects:
No effects on mating performance, fertility or gestation were observed.

Study 3: Maternal toxic effects:no effects
Details on maternal toxic effects:
(a)Signs and mortalities :
At all dosages but in particular at 100 and 300 mg/kg/day, treatment was generally associated with green discolouration of the urine.
There were no other signs of reaction considered attributable to treatment.
Occasional animals were killed, found dead or excluded from the study for reasons of animal welfare. No association with treatment was indicated.
(b)Food consumption :
At 300 mg/kg/day mean food consumption was reduced relative to control values. Differences from control values at 33 and 100 mg/kg/day showed no apparent relationship to dosage.
(c) Bodyweight change :
At 300 mg/kg/day animals showed a slight initial mean weight loss from the start of treatment. Subsequently mean weight gain remained slightly lower than among control animals.
Differences in mean weight gains at 33 and 100 mg/kg/day were not dosage related; overall weight gain was lower than for control animals at 33 mg/kg/day but was slightly higher at 100 mg/kg/day.
(d) Pregnancy rate: Pregnancy rate, as judged by the numbers of animals pregnant at termination, was high in all groups.
(e) Terminal autopsy : There were no macroscopic findings at terminal autopsy that were considered attributable to treatment.

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

clinical signs

dead fetuses

effects on pregnancy duration

maternal abnormalities

mortality

number of abortions

pre and post implantation loss

Remarks on result:

other: Not Specified

Abnormalities:

not specified

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Study 2: Mean offspring weights for treated animals were comparable to controls.
Study 3: no effects observed
Study 4: no effects observed

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Study 2: no effects observed
Study 3: no effects observed
Study 4: no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Study 3: no effects observed
Study 4: no effects observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Study 2: No treatment-related macroscopic abnormalities were detected for the interim death offspring or for the remaining offspring at terminal kill.
Study 3: no effects observed
Study 4: no effects observed

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

Study 3: no effects observed
Study 4: no effects observed
Study 4: no effects observed

External malformations:

effects observed, treatment-related

Description (incidence and severity):

Study 2: Offspring from the 600 mg/kg/day dose group showed less successful completion of surface righting assessments. There were no treatment-related differences in pinna unfolding.
Study 3: no effects observed
Study 4: no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Study 3: Incidences of foetuses with extra thoraco-lumbar ribs or with variant sternebrae showed no statistically significant differences from controls and no relationship to dosage.

Visceral malformations:

no effects observed

Description (incidence and severity):

Study 2: no effects observed
Study 3: no effects observed

Other effects:

not specified

Details on embryotoxic / teratogenic effects:

Study 2: Embryotoxic / teratogenic effects:no effects
Details on embryotoxic / teratogenic effects:
Litter Observations: There were no clinical signs to suggest an effect of treatment.
Offspring Viability: Mean offspring weights for treated animals were comparable to controls.
Offspring Development: Offspring from the 600 mg/kg/day dose group showed less successful completion of surface righting assessments. There were no treatment-related differences in pinna unfolding.
Necropsy of Offspring: No treatment-related macroscopic abnormalities were detected for the interim death offspring or for the remaining offspring at terminal kill.

Study 3: Embryotoxic / teratogenic effects:no effects
Details on embryotoxic / teratogenic effects:
Litter data
(a)Total litter loss – No effect
(b)Litter size, pre and post implantation loss - not affected by the test chemical
(c) Litter and mean foetal weight - not affected by dthe test chemical
(d) Malformations and anomalies - No compound-related fetal malformations or anomalies observable.
At all dosages incidences of visceral and skeletal anomalies appeared unaffected by treatment.
(e) Skeletal variants : Incidences of foetuses with extra thoraco-lumbar ribs or with variant sternebrae showed no statistically significant differences from controls and no relationship to dosage.

Study 4:

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

changes in litter size and weights

external malformations

visceral malformations

Remarks on result:

other: Not Specified

Abnormalities:

not specified

Developmental effects observed:

not specified

Treatment related:

not specified

Conclusions:

Study 2: Based on all the observation and results, the NOAEL for maternal and fetotoxicity study was considered to be 250 mg/Kg bw/day.

Study 3: Based on all the observation and results, it was concluded that, the NOAEL for the maternal animals was 100 mg/kg bw while the NOAEL for the fetal parameters was found to be 300 mg/kg bw.

Study 4: Based on all the observations and results, it was concluded that the NOAEL was considered to be 250 mg/kg/day when Crl:CD (SD) male and female rat were treated with the test chemical.

Executive summary:

Developmental Toxicity Study:

The summaries for the developmental toxicity studies are as follows:

Developmental Toxicity Study 2:

In the combined repeated-dose/reproductive/developmental toxicity screening study, the test material was administered by gavage to three groups each of ten male and ten female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for up to fifty-four consecutive days, at dose levels of 50, 250 and 600 mg/kg/day. The following was recorded for each female: Date of mating, Date and time of observed start of parturition, Date and time of observed completion of parturitionand Duration of gestation. After dosing, it was observed that,Females treated with 600 mg/kg/day showed a higher percentage of pre-implantation losses in comparison to controls, resulting in the birth of less offspring per litter and lower total litter weights at this dose level. Thus, no adverse effects (NOAEL) on mating performance, fertility or gestation were observed at 250 mg/kg-bw/day of concentartion. Also, the same NOAEL was considered for fetal parameters. Thus, based on all the observation and results, it was concluded that the NOAEL of the test chemical for the maternal and fetal parameters considered to be 250 mg/kg bw after administration to the test animals.

Developmental Toxicity Study 3:

The effects of the test chemical on pregnancy of theNew Zealand Whiterabbit were assessed in this study. Daily dosages of 0, 33, 100 or 300 mg of the test chemical. were administered by intragastic intubation during Days 7 to 19 inclusive of pregnancy. The maternal animals were observed forClinical observations, wherein, all animals were regularly handled and observed daily;obvious changes or signs of reaction to treatment were recorded. Mortalities: Any animals that died or were killed for reasons of animal welfare were weighed and subjected to post mortem examination. Body weight: All rabbits were weighed on Days 1, 7, 9, 11, 15, 20, 24 and 29 of gestation. Food consumption: The food intake of all rabbits was measured from weigh-day to weigh-day. Post-mortem examinations: Yes, Sacrifice on gestation day 29, Organs examined: ovaries and uteri the foetuses were removed by caesarean section and examined for visceral and skeletal anomalies. The fetal prameters were observed forIndividual foetal weight, Sex ratio, Number and distribution of embryonic/foetal deaths (classified as) Early i.e. only placenta visible at termination, Late i.e.both placenta and embryonic remnants visible at termination, Foetal abnormalities, and Visceral and skeletal anomalies.On Day 29,animals were sacrficed, litter values determined and foetuses examined for visceral and skeletal abnormality.There was no significant effect on:litter values, as assessed by litter size, pre- and post implantation loss, litter or mean foetal weights andembryonic and foetal development as assessed by incidence of malformations,anomalies and skeletal variants.

Thus, Based on all the observation and results, it was concluded that, the NOAEL for the maternal animals was 100 mg/kg bw while the NOAEL for the fetal parameters was found to be 300 mg/kg bw.

Developmental Toxicity Study 4:

In a combined repeated dose and reproduction/developmental screening test, male and female Crl:CD (SD) rats were treated orally with the test chemical in concentration of 0, 25, 75 or 250 mg/kg/day with recovery dose group of 0 and 250 mg/kg/day.No effect were observe on clinical sign, body weight and food consumption of treated rats as compared to control. Similarly, no effects were observed on blood chemistry, organ weight and histopathology of treated rats as compared to control. In addition, changes were observed as tendency to prolongation in the PT and prolongation in the APTT in male rats at 75 and 250 mg/kg/day dose group and decrease in the RBC and MCHC. An increase in the RET at 250 mg/kg/day dose group during recovery in female rats as compared to control. No reproductive effect were observed in treated male or female rats and on offspring’s as compared to control. Therefore,NOAEL for the parental generation and the F1 generation were considered to be 250 mg/kg/day when male and female Crl:CD (SD) rats were treated orally by gavage with the test chemical.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The data is from a Klimisch 2 database and provides a robust study summary.

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

Developmental Toxicity Study:

The summaries for the developmental toxicity studies are as follows:

Developmental Toxicity Study 2:

In the combined repeated-dose/reproductive/developmental toxicity screening study, the test material was administered by gavage to three groups each of ten male and ten female Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, for up to fifty-four consecutive days, at dose levels of 50, 250 and 600 mg/kg/day. The following was recorded for each female: Date of mating, Date and time of observed start of parturition, Date and time of observed completion of parturitionand Duration of gestation. After dosing, it was observed that,Females treated with 600 mg/kg/day showed a higher percentage of pre-implantation losses in comparison to controls, resulting in the birth of less offspring per litter and lower total litter weights at this dose level. Thus, no adverse effects (NOAEL) on mating performance, fertility or gestation were observed at 250 mg/kg-bw/day of concentartion. Also, the same NOAEL was considered for fetal parameters. Thus, based on all the observation and results, it was concluded that the NOAEL of the test chemical for the maternal and fetal parameters considered to be 250 mg/kg bw after administration to the test animals.

Developmental Toxicity Study 3:

The effects of the test chemical on pregnancy of theNew Zealand Whiterabbit were assessed in this study. Daily dosages of 0, 33, 100 or 300 mg of the test chemical. were administered by intragastic intubation during Days 7 to 19 inclusive of pregnancy. The maternal animals were observed forClinical observations, wherein, all animals were regularly handled and observed daily;obvious changes or signs of reaction to treatment were recorded. Mortalities: Any animals that died or were killed for reasons of animal welfare were weighed and subjected to post mortem examination. Body weight: All rabbits were weighed on Days 1, 7, 9, 11, 15, 20, 24 and 29 of gestation. Food consumption: The food intake of all rabbits was measured from weigh-day to weigh-day. Post-mortem examinations: Yes, Sacrifice on gestation day 29, Organs examined: ovaries and uteri the foetuses were removed by caesarean section and examined for visceral and skeletal anomalies. The fetal prameters were observed forIndividual foetal weight, Sex ratio, Number and distribution of embryonic/foetal deaths (classified as) Early i.e. only placenta visible at termination, Late i.e.both placenta and embryonic remnants visible at termination, Foetal abnormalities, and Visceral and skeletal anomalies.On Day 29,animals were sacrficed, litter values determined and foetuses examined for visceral and skeletal abnormality.There was no significant effect on:litter values, as assessed by litter size, pre- and post implantation loss, litter or mean foetal weights andembryonic and foetal development as assessed by incidence of malformations,anomalies and skeletal variants.

Thus, Based on all the observation and results, it was concluded that, the NOAEL for the maternal animals was 100 mg/kg bw while the NOAEL for the fetal parameters was found to be 300 mg/kg bw.

Developmental Toxicity Study 4:

In a combined repeated dose and reproduction/developmental screening test, male and female Crl:CD (SD) rats were treated orally with the test chemical in concentration of 0, 25, 75 or 250 mg/kg/day with recovery dose group of 0 and 250 mg/kg/day.No effect were observe on clinical sign, body weight and food consumption of treated rats as compared to control. Similarly, no effects were observed on blood chemistry, organ weight and histopathology of treated rats as compared to control. In addition, changes were observed as tendency to prolongation in the PT and prolongation in the APTT in male rats at 75 and 250 mg/kg/day dose group and decrease in the RBC and MCHC. An increase in the RET at 250 mg/kg/day dose group during recovery in female rats as compared to control. No reproductive effect were observed in treated male or female rats and on offspring’s as compared to control. Therefore,NOAEL for the parental generation and the F1 generation were considered to be 250 mg/kg/day when male and female Crl:CD (SD) rats were treated orally by gavage with the test chemical.

Justification for classification or non-classification

Based on the summaries of the available data for toxicity to reproduction as well as developmental toxicity, it is considered that the test chemical might not qualify for classification as a reproductive or developmental toxicant.