Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: Screening test
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 June 2012 to 27 August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed in accordance with OECD & US EPA test guidelines in compliance with GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
GLP compliance:
yes
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
6,6'-di-tert-butyl-4,4'-butylidenedi-m-cresol
EC Number:
201-618-5
EC Name:
6,6'-di-tert-butyl-4,4'-butylidenedi-m-cresol
Cas Number:
85-60-9
Molecular formula:
C26H38O2
IUPAC Name:
6,6'-di-tert-butyl-4,4'-butylidenedi-m-cresol
Constituent 2
Reference substance name:
6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol
IUPAC Name:
6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Identification: 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol
Molecular formula: C26H38O2
Molecular weight: 382.58
CAS Number: 85-60-9
Description: White powder
Batch: W4B9F0001
Purity: 99.4%
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 31 May 2013
Hygroscopic: No
Volatile: No
pH: Not indicated
Stability at higher temperatures: Not indicated
Stability in vehicle (Propylene glycol): Unknown
Solubility in vehicle (Propylene glycol): Not indicated

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test system Rat: Crl:WI(Han) (outbred, SPF-Quality). Nulliparous and non-pregnant females and untreated animals were used at initiation of the study.
Rationale: This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. WIL Research Europe B.V. has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Source F0: Charles River Deutschland, Sulzfeld, Germany.
Age at start F0-treatment: Approximately 11 weeks.
Number of F0-animals: 40 females and 40 males.
Acclimatization F0: At least 5 days prior to start of treatment.
Health inspection F0: Upon receipt of the animals.
Randomization F0: Prior to commencement of treatment, by computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Identification F0: Earmark and tattoo.
Identification of pups: On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink.
Room number: A0.12.
Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
Accommodation
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm).
General: Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied.
Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
Water: Free access to tap-water.
Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on exposure:
Vehicle: Propylene glycol, specific gravity 1.036 (Merck, Darmstadt, Germany).
Rationale for vehicle: Based on trial formulations performed at WIL Research Europe B.V.
Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity/density of the vehicle. No correction was made for the purity/composition of the test substance.
Storage conditions: At ambient temperature.
Details on mating procedure:
Mating procedures: Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating occurred, the males and females were separated. Detection of mating was not confirmed for animal no. 61 which did deliver live offspring. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum.
Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes and placentas cleaned up, nest build up and/or feeding of pups started). Females that were littering were left undisturbed.
Number of pups: 470 pups.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on two occasions during the treatment phase Day 6 (09 July 2012) and Day 10 (13 July 2012), according to a validated method (Project 499464). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 39-54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Female nos. 53 and 59 (Group 2) were not dosed during littering.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.
Details on study schedule:
After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 100, 300 and 1000 mg/kg/day. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 39-54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
10 male and 10 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Parental animals
Method: Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Pups: Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Positive control:
Postive control not utilised for this study.

Examinations

Parental animals: Observations and examinations:
Mortality / Viability: At least twice daily. The circumstance of any death was recorded in detail.
Clinical signs: Daily, detailed clinical observations were conducted for all animals and were started at least within 30 minutes after dosing (on the peak period of anticipated effects after dosing).
The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.
Body weights: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.
Food consumption: Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on Days 1 and 4 of lactation.
Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.
General reproduction data: Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.
Oestrous cyclicity (parental animals):
Not specified.
Sperm parameters (parental animals):
Not specified.
Litter observations:
Each litter was examined to determine the following, if practically possible:
Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were conducted for all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: Sex was determined for all pups on Days 1 and 4 of lactation.
Postmortem examinations (parental animals):
The animals were not deprived of food overnight. Animals surviving to scheduled necropsy were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated.

All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
The numbers of former implantation sites and corpora lutea were recorded for all paired females.
These numbers were not reported for non-pregnant and non-mated females.

Organ weights: The following organ weights and terminal body weight were recorded from all F0-males on the scheduled day of necropsy:
Epididymides and Testes

Histopathology
A peer review on the histopathology data was performed by a second pathologist.
The following slides were examined by a pathologist:
- The ovaries, testes and epididymides of the animals of Groups 1 and 4.
- The additional slides of the testes of the males of Groups 1 and 4, and all males suspected to be infertile or which died before mating, to examine staging of spermatogenesis.
- The preserved organs and tissues of the animal (no. 67) which died spontaneously.
- All gross lesions of all animals (all dose groups).
- The reproductive organs* of all males that failed to sire (Group 2: no. 16, Group 3 no. 29) and all females that failed to deliver healthy pups (Group 2: no. 56, Group 3: no. 69 and no. 67 who died during delivery). Additionally, the reproductive organs were examined for male no. 27 with whom no. 67 was paired.
* Reproductive organs includes the cervix, clitoral gland, coagulation gland, epididymides, ovaries, preputial gland, prostate gland, seminal vesicles, testes, uterus, and vagina.
Postmortem examinations (offspring):
Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation.
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
Not specified.
Offspring viability indices:
Not specified.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female (no. 67) at 300 mg/kg died during delivery. Subsequent examination could not determine a cause of death that was related to treatment. This was considered to be incidental in nature and not related to treatment.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

Mortality: No mortality occurred during the study period that was considered to be related to treatment with the test substance.
One female (no. 67) at 300 mg/kg died during delivery. Subsequent examination could not determine a cause of death that was related to treatment. This was considered to be incidental in nature and not related to treatment.

Clinical signs: No clinical signs of toxicity were noted during the observation period.
Alopecia was noted for a single female at 100 mg/kg (no. 52). This was incidental in nature.

Body weights: Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

Food consumption: Food consumption before or after allowance for body weight was similar between treated and control animals up to 1000 mg/kg.

Macroscopic examination: Necropsy did not reveal any alterations that were attributable to treatment up to 1000 mg/kg.
Female no. 67 who died during delivery was noted with dilation and a prolapsed vagina, a greenish, soft nodule on the right clitoral gland and 2 fetuses were found in the right uterine horn.
Incidental findings included pelvic dilation of the right kidney, alopecia of the shoulder and abdominal region, reddish hard nodule on the left uterine horn and vagina contains reddish fluid. At the limited incidence observed, these were not considered to be toxicologically relevant.

Organ weights: There were no toxicologically relevant effects on testes and epididymides weights and terminal body weights up to 1000 mg/kg.
The testes to body weight ratio was significantly lower for males at 300 mg/kg. This was mainly attributable to the low ratio for animal no. 28 and was not considered to be related to treatment.

Microscopic examination: There were no treatment related microscopic findings.
A definitive cause of death could not be established for female no. 67. The nodules in the uterus of animal no. 58 seen at the macroscopic examination represented implantation sites.
There were no microscopic findings in any of the animals suspected of infertility which could explain their lack of reproductive performance.
The remaining recorded microscopic findings were within the range of background pathology encountered in Wistar (Han) rats of this age in this type of study and occurred at similar incidences and severity in both control and treated rats. The spermatogenic staging profiles were normal for all males evaluated.

Reproduction data: No toxicologically relevant effects on reproductive parameters were noted. There were 10, 9, 9 and 10 pregnant females in the control, 100, 300 and 1000 mg/kg groups, respectively.
The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects clinical signs; mortality; body weight; food consumption; water consumption; gross pathology; organ weights; histopathology;

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Pale or lean appearance, no milk in the stomach and red spot on the head were incidental clinical signs noted. The nature and limited occurrence of these clinical signs remained within the range considered normal for pups of this age, and were not considered toxicologically relevant
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Three pups of the control group and two, two and eight pups of the 100, 300 and 1000 mg/kg groups, respectively were found dead or went missing during the first days of lactation. Missing pups were most likely cannibalized. Seven of the eight dead pups at 1000 mg/kg were all from female no. 80 who had three living and seven dead pups at first litter check. In the absence of any adverse effects seen for any other female in at this dose level, it was not considered to be treatment related. No toxicological relevance was attributed to any of these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not specified
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
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Incidental macroscopic findings of pups that were found dead included no milk in the stomach and cannibalism of the whole body except the hind legs (noted for three of the seven dead pups from female no. 80). There were no macroscopic findings noted for surviving pups. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant
Histopathological findings:
no effects observed
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

Mortality: Three pups of the control group and two, two and eight pups of the 100, 300 and 1000 mg/kg groups, respectively were found dead or went missing during the first days of lactation. Missing pups were most likely cannibalized. Seven of the eight dead pups at 1000 mg/kg were all from female no. 80 who had three living and seven dead pups at first litter check. In the absence of any adverse effects seen for any other female in at this dose level, it was not considered to be treatment related. No toxicological relevance was attributed to any of these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

Clinical signs: Pale or lean appearance, no milk in the stomach and red spot on the head were incidental clinical signs noted. The nature and limited occurrence of these clinical signs remained within the range considered normal for pups of this age, and were not considered toxicologically relevant.

Body weights: Body weights of pups were unaffected by treatment up to 1000 mg/kg.

Macroscopy: Incidental macroscopic findings of pups that were found dead included no milk in the stomach and cannibalism of the whole body except the hind legs (noted for three of the seven dead pups from female no. 80). There were no macroscopic findings noted for surviving pups. The nature and incidence of these findings remained within the range considered normal for pups of this age, and were therefore not considered to be toxicologically relevant.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects noted.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

BODY WEIGHTS (GRAM) SUMMARY

MALES

 

GROUP 1 CONTROL

GROUP 2

100 MG/KG

GROUP 3 300 MG/KG

GROUP 4

1000 MG/KG

PRE MATING

DAY 1

WEEK 1

MEAN

314

317

318

315

ST.DEV

4.3

12.5

12.2

10.8

N

10

10

10

10

DAY 8

WEEK 2

MEAN

328

333

335

328

ST.DEV.

7.1

15.0

18.8

14.1

N

10

10

10

10

MATING PERIOD

DAY 1

WEEK 1

MEAN

342

349

351

342

ST.DEV

8.1

15.1

22.6

17.6

N

10

10

10

10

DAY 8

WEEK 2

MEAN

352

357

362

352

ST.DEV.

10.2

16.1

25.4

18.2

N

10

10

10

10

DAY 15

 WEEK 3

MEAN

366

368

376

366

ST.DEV.

11.7

19.3

26.8

20.5

N

10

10

10

10

FEMALES

PRE MATING

DAY 1

WEEK 1

MEAN

208

212

210

209

ST.DEV

5.4

7.3

6.7

5.4

N

10

10

10

10

DAY 8

WEEK 2

MEAN

218

223

222

217

ST.DEV.

5.4

8.8

6.6

6.5

N

10

10

10

10

MATING PERIOD

DAY 1

WEEK 1

MEAN

228

233

229

228

ST.DEV.

9.0

9.0

7.7

7.6

N

10

10

10

10

DAY 8

WEEK 2

MEAN

 

247

256

 

ST.DEV.

 

---

7.1

 

N

 

1

2

 

DAY 15

WEEK 3

MEAN

 

 

278

 

ST.DEV.

 

 

14.8

 

N

 

 

2

 

DAY 22

WEEK 4

MEAN

 

 

305

 

ST.DEV.

 

 

74.2

 

N

 

 

2

 

DAY 29

WEEK 5

MEAN

 

 

253

 

ST.DEV.

 

 

---

 

N

 

 

1

 

 

BODY WEIGHTS (GRAM) SUMMARY – FEMALES F0-GENERATION

 

GROUP 1 CONTROL

GROUP 2

100 MG/KG

GROUP 3

300 MG/KG

GROUP 4

1000 MG/KG

POST COITUM

DAY 0

MEAN

230

238

234

230

ST.DEV.

7.7

9.4

9.9

7.5

N

10

9

8

10

DAY 4

MEAN

245

254

249

246

ST.DEV.

9.1

10.1

11.1

7.3

N

10

9

8

10

DAY 7

MEAN

253

261

256

254

ST.DEV.

9.2

11.7

13.1

8.9

N

10

9

8

10

DAY 11

MEAN

270

278

274

271

ST.DEV.

10.2

12.2

15.1

10.6

N

10

9

8

10

DAY 14

MEAN

284

291

287

286

ST.DEV.

11.5

13.1

14.8

11.5

N

10

9

8

10

DAY 17

MEAN

309

315

311

311

ST.DEV.

13.3

14.0

16.7

15.2

N

10

9

8

10

DAY 20

MEAN

347

355

350

351

ST.DEV.

13.9

18.1

21.9

15.1

N

10

9

8

10

LACTATION

DAY 1

MEAN

268

275

269

272

ST.DEV.

11.9

13.8

13.0

10.9

N

10

9

8

10

DAY 4

MEAN

280

286

281

286

ST.DEV.

12.0

16.5

14.4

14.6

N

10

9

8

10

 

MACROSCOPIC FINDINGS SUMMARY

MALES

 

GROUP 1 CONTROL

GROUP 2

100 MG/KG

GROUP 3

300 MG/KG

GROUP 4

1000 MG/KG

END OF TREATMENT

Animals examined

10

10

10

10

Animals without findings

10

10

10

9

Animals affected

0

0

0

1

Kidneys

Pelvic dilation

0

0

0

1

FEMALES

INTERCURRENT DEATH

Animals examined

 

 

1

 

Animals affected

 

 

1

 

Uterus

Contents:

 

 

1

 

Vagina

Prolapsed

 

 

1

 

Dilation

 

 

1

 

END OF TREATMENT

Animals examined

10

10

9

10

Animals without findings

10

8

8

10

Animals affected

0

2

1

0

Uterus

Nodule(s)

0

1

0

0

Vagina

Contains fluid

0

1

0

0

Clitoral glands

 

 

 

 

Nodule(s)

0

0

1

0

Skin

Alopecia

0

1

0

0

 

REPRODUCTION DATA SUMMARY

 

GROUP 1 CONTROL

GROUP 2

100 MG/KG

GROUP 3

300 MG/KG

GROUP 4

1000 MG/KG

Females paired

10

10

10

10

Females mated

10

10

9

10

Females pregnant

10

9

9

10

Non-pregnant females

0

1

0

0

Non-mated females

0

0

1

0

Females with living pups on Day 1

10

9

9

10

Females dead during delivery

0

0

1

0

Mating index (%)

100.0

100.0

90.0

100.0

(Females mated / Females paired) * 100

Fertility index (%)

100.0

90.0

90.0

100.0

(Pregnant females / Females paired) * 100

Conception index (%)

100.0

90.0

100.0

100.0

(Pregnant females / Females mated) * 100

Gestation index (%)

100.0

100.0

100.0

100.0

(Females with living pups on Day 1 / Pregnant females ) * 100

 

DEVELOPMENTAL DATA F0-GENERATION - LACTATION

 

GROUP 1 CONTROL

GROUP 2

100 MG/KG

GROUP 3 300 MG/KG

GROUP 4 1000 MG/KG

LITTERS

TOTAL

10

9

8

10

DURATION OF GESTATION

MEAN (+)

21.4

21.3

21.4

21.6

ST.DEV.

0.7

0.5

0.5

0.5

N

10

9

8

10

DEAD PUPS AT FIRST LITTER CHECK

LITTERS AFFECTED (#)

1

0

0

1

TOTAL

1

0

0

7

MEAN (+)

0.1

0.0

0.0

0.7

ST.DEV.

0.3

0.0

0.0

2.2

N

10

9

8

10

LIVING PUPS AT FIRST LTTER CHECK

% OF MALES / FEMALES (#)

50 / 50

44 / 56

47 / 53

45 / 55

TOTAL

118

111

102

120

MEAN (+)

11.8

12.3

12.8

12.0

ST.DEV.

1.1

2.0

2.7

3.4

N

10

9

8

10

POSTNATAL LOSS

% OF LIVING PUPS

1.7

1.8

2.0

0.8

LITTERS AFFECTED (#)

2

2

2

1

TOTAL (#)

2

2

2

1

MEAN (+)

0.2

0.2

0.3

0.1

ST.DEV.

0.4

0.4

0.5

0.3

N

10

9

8

10

VIABILITY INDEX (#)

98.3

98.2

98.0

99.2

 

BODY WEIGHTS OF PUPS (GRAM) F0-GENERATION - LACTATION

DAY

SEX

 

GROUP 1 CONTROL

GROUP 2 100 MG/KG

GROUP 3 300 MG/KG

GROUP 4 MG/KG

1

M

MEAN

6.6

6.4

6.5

6.6

 

 

ST.DEV.

0.5

0.6

0.8

0.5

 

 

N

10

9

8

10

 

F

MEAN

6.3

6.1

6.2

6.3

 

 

ST.DEV.

0.4

0.6

0.6

0.4

 

 

N

10

9

8

9

 

M+F

MEAN

6.5

6.2

6.4

6.4

 

 

ST.DEV.

0.5

0.6

0.6

0.4

 

 

N

10

9

8

10

4

M

MEAN

9.9

9.6

9.8

10.3

 

 

ST.DEV.

0.9

1.1

1.3

1.0

 

 

N

10

9

8

10

 

F

MEAN

9.5

9.3

9.4

9.7

 

 

ST.DEV.

0.8

1.3

1.1

0.7

 

 

N

10

9

8

9

 

M+F

MEAN

9.8

9.4

9.6

10.1

 

 

ST.DEV.

0.9

1.2

1.1

1.0

 

 

N

10

9

8

10

Applicant's summary and conclusion

Conclusions:
Based on these results, a parental, reproductive and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg/day was derived.
Executive summary:

Title: Reproduction/developmental toxicity screening test of 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol in rats by oral gavage.

 

Guidelines: The study was based on the following guidelines:

1) OECD 421, Reproduction/Developmental Toxicity Screening Test, July 1995.

2) OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.

 

Rationale for dose levels: Based on the results of a 10-day dose range finding study (Project 499462), the dose levels for this reproduction/developmental toxicity screening test were 100, 300 and 1000 mg/kg.

 

Study outline: After acclimatization, four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 100, 300 and 1000 mg/kg/day. Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 39-54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

 

Evaluated parameters: The following observations and examinations were evaluated: mortality / viability, clinical signs (daily), body weight and food consumption (at least at weekly intervals), macroscopy at termination, organ weights and histopathology on a selection of tissues, and reproduction/developmental parameters, consisting of mating, fertility and conception indices, precoital time, number of corpora lutea and implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights and macroscopy).

Formulations were analyzed twice during the study to assess accuracy, homogeneity and stability.

 

Results/discussion: The accuracy, homogeneity and stability results were outside the acceptable range during the first formulation analysis. Subsequent analyses revealed the formulations were prepared accurately and were considered stable for at least 6 hours at room temperature, though formulations were not homogenous. The results were attributable to an interaction between limited sensitivity of the analytical method and the nature of the test substance itself and were ultimately accepted. The inhomogeneity of the test substance had no negative impact on the study as formulations were stirred constantly during dosing.

Parental results: No parental toxicity was observed up to the highest dose level tested (1000 mg/kg).

Reproductive results: No reproductive toxicity was observed up to the highest dose level tested (1000 mg/kg).

Developmental results: No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg).

 

Conclusion: Treatment with 6,6’-di-tert-butyl-4,4’-butylidenedi-m-cresol by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg body weight/day revealed no parental, reproductive, or developmental toxicity up to 1000 mg/kg body weight/day.

Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg/day was derived.

Categories Display