6,6'-di-tert-butyl-2,2'-methylenedi-p-cresol

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP and OECD Guideline study (TG 421), acceptable documented (abstract and result tables in English, publication in Japanese)

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1999

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

6,6'-di-tert-butyl-2,2'-methylenedi-p-cresol

EC Number:

204-327-1

EC Name:

6,6'-di-tert-butyl-2,2'-methylenedi-p-cresol

Cas Number:

119-47-1

Molecular formula:

C23H32O2

IUPAC Name:

2-tert-butyl-6-[(3-tert-butyl-2-hydroxy-5-methylphenyl)methyl]-4-methylphenol

Details on test material:

purity: 98.2%, produced by Sumitomo Chemical, Lot No: 710140

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Weight at study initiation: (P) Males: 332-383 g; Females: 206-238 g

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: in 5% gum arabic

Details on exposure:

oral (gavage)

Details on mating procedure:

Male/female percage: 171, length of cohabitation: at the most 14 d, until proof of pregnancy (formation of vaginal closing or sperm detection in vagina)

Duration of treatment / exposure:

male: 50-52 d, female: 40-48 d (from 14 days before mating to the day 3 of lactation)

Frequency of treatment:

daily

Details on study schedule:

age at study initiation was 10 wk old (332-383 g for male, 206-238 g for female)

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 12.5, 50, 200, 800 mg/kg bw and day
Basis:
other: nominal

No. of animals per sex per dose:

12/per dose group/sex

Control animals:

yes, concurrent vehicle

Positive control:

none

Examinations

Parental animals: Observations and examinations:

clinical observations: general appearance twice a day, organ weights: testis, epididymis, cauda epididymis, ovary, microscopic evalauations:( control and all teatment groups): testis, caput epididymis, (control and 800 mg/kg group): seminal vesicle, ovary

Sperm parameters (parental animals):

Parameters examined in male parental generations:
testis weight, epididymis weight, sperm count in testes, sperm count in epididymides, sperm motility, viability, sperm morphology

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals

Statistics:

Dunnett's or Scheffe's test for continuous data and Chi square test for quantal data

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

no effects observed

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

12.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects

2

Dose descriptor:

LOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Giant cell formation in the testes, decreases in sperm motility ratio and number of sperms in the epididymis cauda, increase in abnormal sperm ratio

3

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects

4

Dose descriptor:

LOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: decreased body weight gain, lower food consumption, dreased number of corpora lutea, decreased number of implantation scars and number of pup born

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Mortality / viability:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

Details on results (F1)

NOAEL: female reproductive toxicity: 50 mg/kg bw/daw
NOAEL: male reproductive toxicity: 12.5 mg/kg bw/day
NOAEL developmental toxicity: 50 mg/kg bw/day

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Pups (200 mg/kg bw): Pup numbers on day 0 and 4 of lactation were decreased

Remarks on result:

other: Pups (800 mg/kg bw): the number of pups on day 0 and 4 of lactation, live birth index, and body weights of both sexes on day 4 of lactation were decreased, and the number of stillbirths was increased

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Parental animals:

Males and females: no mortality and clinical signs recorded.

Males (50 mg/kg bw): Giant cell formation in the testes, decreases in sperm motility ratio and number of sperms in the epididymis cauda, and an increase in abnormal sperm ratio

Males (200 mg/kg bw): Atrophy of the testes and epididymides, decreases in the absolute and relative testis and epidymis weights, atrophy of seminiferous tubules, degeneration of seminiferous tubules, a decrease in sperm in the epididymis cauda and giant cell formation were noted in the testes, a decrease in the sperm motility ratio and an increase in the abnormal sperm ratio

Males (800 mg/kg bw): A transient decrease in food consumption, atrophy of the testes, epididymides and seminal vesicles, decreases in the absolute and relative testis and epididymis weights, atrophy of seminiferous tubules in the testes, on sperm examination: no motile sperm werenoted, the number of abnormal sperm tended to increase, and the number of sperm in the epididymis cauda were decreased

Females (200 mg/kg bw): Supression of body weight gain during the lactation period, lower food consumption was evident during the pre-mating, pregnancy and lactation periods, decrease in the number of corpora lutea, decrease in number of implantation scars and number of pups born, 1 dam was unable to deliver pups, and 1 dam lost all their pups during the lactation period

Females (800 mg/kg bw): Supression of body weight gain was noted during the pregnancy and lactation periods, lower food consumption was noted during the pre-mating, pregnancy and lactation periods; decrease in the number of corpora lutea, decrease in number of implantation scars and number of pups born, 1 dam was unable to deliver pups, and 1 dam lost all their pups during the lactation period

Offspring:

Pups (200 mg/kg bw): Pup numbers on day 0 and 4 of lactation were decreased

Pups (800 mg/kg bw): the number of pups on day 0 and 4 of lactation, live birth index, and body weights of both sexes on day 4 of lactation were decreased, and the number of stillbirths was increased

Table body weights parental animals

Male (number)	12	12	12	12	12
Dose level (mg/kg/day)	0	12.5	50	200	800
Body weight (g)	535.8 ± 41.7	536.5 ± 33.5	544.3 ± 31.4	539.7 ± 35.7	514 ± 34.4
Female (number)	12	12	12	12	12
Body weight (g)	310.4 ± 12.3	312.2 ± 18.9	310.7 ± 17.2	287.4 ± 13.3**	281.9 ± 22.9**

Significantly different from control (**:p<0.01)

Testis and epididymis weights in male

Male (number)	12	12	12	12	12
Dose level (mg/kg/day)	0	12.5	50	200	800
Testis absolute wt. (g, Mean ± SD)	3.550 ± 0.333	3.598 ± 0.320	3.558 ± 0.302	2.983 ± 0.767*	1.736 ± 0.263**
Testis relative wt. (g%, Mean ± SD)	0.666 ± 0.082	0.674± 0.072	0.655 ± 0.046	0.557 ± 0.157*	0.338 ± 0.050**
Epididymis absolute wt. (g, Mean ± SD)	1.255 ± 0.143	1.343 ± 0.118	1.196 ± 0.113	1.108 ± 0.125*	0.924 ± 0.100**
Epididymis relative wt. (g%, Mean ± SD)	0.235 ± 0.034	0.250± 0.024	0.220 ± 0.018	0.205 ± 0.027*	0.180 ± 0.020**

(* p<0.05, **p<0.01)

Histopathological changes in testis and epididymis male

Dose level (mg/kg/day)	0	12.5	50	200	800
Testis
Atrophy, seminiferous tuble	0/12	0/12	0/12	6/12**	12/12**
Degeneration, seminiferous tuble	0/12	0/12	0/12	1/12	0/12
Decrease, sperm	0/12	0/12	0/12	1/12	0/12
Giant cell formation	0/12	0/12	2/12	2/12	0/12
Epididymis
Decrease, sperm	0/12	0/12	0/12	9/12**	12/12**

( * p<0.05, ** p<0.01)

Sperm abnormality in male

Dose level (mg/kg/day)	0	12.5	50	200	800
Sperm motion parameters
After 30 min. incubation
Motility ratio (%)	71.96 ±9.69	74.92 ± 7.81	60.42± 10.26**	14.50 ± 21.75**	0.00 ± 0.00**
Curvilinear velocity (um/s)	348.95 ±20.87	369.08 ± 16.17*	364.94± 18.14	301.08 ± 104.59	-
Bear cross frequency (Hz)	30.64 ±1.77	30.16 ± 1.59	32.91± 1.70**	29.98 ± 10.51*	-
Morphology of sperm
Abnormal ratio (%)	1.55 ±3.63	0.55 ± 0.55	8.11± 6.33**	56.33 ± 29.03**	-
Viabity (%)	98.57 ±2.04	99.56 ± 0.47	89.19± 11.47**	71.68 ± 9.31**	-
Survivability (%)	83.29 ±6.87	86.44 ± 3.27	66.03± 17.79**	39.03 ± 15.16**	-
Number of sperms (left epididymis cauda x10^6)	207.41 ±60.16	222.42 ± 49.26	128.00± 39.88**	60.73 ± 29.17*	-
Number of sperms/g (left epididymis cauda x10^6)	707.41±153.02	704.85 ± 154.64	503.29± 159.44**	238.88 ± 102.42**	-

(*p <0.05, **p<0.01)

Table: Organ weights of female rats

Dose level (mg/kg/day)	0	12.5	50	200	800
Number of dams	12	12	12	12	12
Ovaries (mg)	94.50 ± 12.06	91.43 ± 10.00	89.88 ± 8.77	89.69 ± 16.74	88.78 ± 14.65
Ovaries (mg%)	30.51 ± 4.20	29.36 ± 3.40	28.90 ± 1.90	31.23 ± 5.75	31.44 ± 3.93

Number of estrous cases and reproductive performance

Dose (mg/kg)	0	12.5	50	200	800
Number of females	12	12	12	12	12
Number of estrous cases before mating (14 days)	3.3 ± 0.5	3.5 ± 0.5	3.5 ± 0.5	3.7 ± 0.5	3.2 ± 0.6
Fertility index (%)	100.0	100.0	100.0	91.7	100.0
number of pregnant females with live pups	12	12	12	11	10

Fertility/Developmental toxicity

Dose level (mg/kg day)	0	12.5	50	200	800
No. of pairs mated	12	12	12	12	12
No. of pregnant females	12	12	12	12	12
Corpora lutea	16.4 ± 3.0	16.4 ± 2.6	16.3 ± 1.5	15.1 ± 1.4	14.1 ± 1.6*
Implantation scars	14.3 ± 3.0	14.7 ± 1.1	15.2 ± 1.3	13.5 ± 1.4	13.1 ± 1.5*
Pups born	13.5 ± 3.3	13.5 ± 1.0	14.8 ± 1.3	11.7 ± 1.4**	12.2 ± 1.8*
Delivery Index (%)	93.5 ± 8.9	92.2 ± 5.2	97.3 ± 3.3	87.2 ± 10.5*	92.8 ± 5.7
Live pups	13.1 ± 3.2	13.3 ± 0.8	14.3 ±1.4	11.4± 1.0**	12.4 ± 1.8
Dead pups on day 0 of lactation	0.4 ± 0.7	0.2 ± 0.4	0.4 ± 0.5	0.2 ± 0.6	0.9 ± 2.7
Live birth index (%)	97.1 ± 4.6	98.9 ± 2.5	97.2 ± 3.5	98.8 ± 3.9	89.9 ± 3.0
Live pups on day 4 of lactation	13.1 ± 3.2	13.3 ± 0.8	14.3 ±1.4	11.4 ± 1.0**	12.4 ± 1.8
Body weight of live pups (g) on day 0 Males	6.78 ± 0.40	6.81 ± 0.23	6.90 ± 0.50	7.60 ± 0.52**	6.97 ± 0.71
Body weight of live pups (g) on day 0 Females	6.43 ± 0.37	6.40 ± 0.17	6.53 ± 0.44	7.25 ± 0.49**	6.61 ± 0.66
Body weight of live pups (g) on day 4 Males	11.02± 0.83	11.05 ± 0.77	10.58 ± 1.11	11.29 ± 1.14	9.68 ± 1.72*
Body weight of live pups (g) on day 4 Females	10.33 ± 0.81	10.36 ± 0.67	10.20 ± 1.15	10.77 ± 1.04	9.30 ± 1.64

* p<0.05, ** p<0.01)

Applicant's summary and conclusion

Executive summary:

In a reproduction/developmental toxicity screening test (OECD Guideline 421) with rats toxic effects of the test substance Vulcanox BKF were seen. Male and females Crj:CD rates were treated with 0, 12.5, 50, 200 and 800 mg/kg bw/day test substance. In males food consumption was decreased transiently in the 800 mg/kg bw/day group. An atrophy of the testes and epididymides was noted at 200 mg/kg bw/ day and higher. In addition, atrophy of the seminal vesicles was found at 800 mg/kg bw/day. Moreover a decrease in the absolute and relative testis and epididymis weights were noted at 200 and 800 mg/kg bw/ day. A decrease in the sperm motility ratio, sperm viability ratio, sperm survivability ratio, and number of sperm in the epididymis cauda was indicated at concentration of 50 and 200 mg/kg bw/day; in addition, an increase in the abnormal sperm ratio was seen at these concentrations. At the highest dose group (800 mg/kg bw/day), no motile sperm were evident and the number of abnormal sperm tended to increase. Moreover, the total number of sperm was decreased. Histological examinations revelated giant cell formation in the testes at 50 mg/kg bw/day and higher. Atrophy and degeneration of the seminiferous tubules were noted at 200 mg/kg bw/day and at 800 mg/kg bw/day an atrophy of seminiferous tubules was found. In female rats a suppression of the body weight gain was noted in the 200 mg/kg bw/day treatment group during the lactation period. In the highest dose group (800 mg/kg bw/day) a suppression of body weight gain was seen during the pregnancy and lactation periods. Lower food consumption was noted in the 200 and 800 mg/kg bw/day groups during pre-mating, pregnancy, and lactating periods. Decreases in the number of corpora lutea, number of implantation scars, and number of pups born were noted at 200 and 800 mg/kg bw/day. In addition, 1 dam was unable to deliver pups, and 1 dam lost all their pups during the lactation period at 800 mg/kg bw/day. Based on the findings of this study, the NOAEL for reproductive toxicity is assessed to be 50 mg/kg bw/day for females and 12.5 mg/kg bw/day for males. The NOAEL for pup development is considered to be 50 mg/kg bw/day (MHWJ 1999).