Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
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)
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
Weight at study initiation: (P) Males: 332-383 g; Females: 206-238 g
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)
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
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
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
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
effects observed, treatment-related
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
no effects observed
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
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
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
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
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
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
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
Reproductive effects observed:
not specified

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 x106)  207.41 ±60.16  222.42 ± 49.26  128.00± 39.88**  60.73 ± 29.17*

-

Number of sperms/g (left epididymis cauda x106)  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) 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)

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).

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
12.5 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
OECD TG 421
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a reproduction/developmental toxicity screening test (OECD Guideline 421) with rats toxic effects of the test substance 6,6’-di-tert-butyl-2,2’-methylendi-p-cresol (DBMC) were seen. Male and females Sprague-Dawley rats were treated per gavage with 0, 12.5, 50, 200 and 800 mg/kg bw/day test substance. No mortality and clinical signs were noted. 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, an 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 was evident and the number of abnormal sperm tended to increase. Moreover, the total number of sperm was decreased. Histological examinations revealed 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 period. A slight decrease in the number of corpora lutea (200 mg/kg: 15.1 ± 1.4, 800 mg/kg: 14.1 ±1.6, control: 16.4 ± 3.0), number of implantation scars (200 mg/kg bw:13.5 ± 1.4, 800 mg/kg 13.1 ± 1.5, control: 14.3 ± 3.0), and number of pups born (200 mg/kg bw: 11.7 ± 1.4, 800 mg/kg: 12.2 ± 1.8, control: 13.5 ± 3.3) 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 (MHWJ 1999). Based on the findings of this study, the NOAEL for systemic and reproductive toxicity is assessed to be 50 mg/kg bw/day for females and 12.5 mg/kg bw/day for males.

In a two month feeding study with male Fischer rats the test substance 6,6’-di-tert-butyl-2,2’-methylendi-p-cresol (DBMC) induced toxic effects in the testis. Animals feed with 0.06% test substance (approx. 40 to 60 mg/kg/day) showed a decrease in relative testicular and epidididymal weights and histopathological changes like vacuolisation of Sertoli cells, disappearance of basement membrane and degeneration of spermatids. Moreover, the daily sperm production (DSP) was significant decreased in treated animals; whereas the serum testosterone levels were not significant changed in treated animals compared to control. In addition, no estrogenic activity of the test substance was noted in an in vitro ERa-binding assay (Takahashi 2006).

This working group (Takahashi et al.) also evaluated mice in a two months feeding study. Male Crj:CD-1 (ICR) mice were feed with 0.25% test substance (approx. 414 mg/kg bw/day). No significant changes in absolute organ weights (testes, accessory organ weights, liver and kidney) were found after treatment with the test substance in male mice. However, treated animals showed histopathological changes like giant cells (6/8) and Leidig cell vacuolization (2/8) (Takahashi 2006).

In additions, in a subacute feeding study with Sprague-Dawley rat (MHWJ 1996) adverse effects on the testis of treated males were noted. Male and female Sprague Dawley rats (six per dose and sex) were treated per gavage for 28 days with the test substance at concentration of 0, 50, 200, 800 mg/kg bw/day. A 14-day recovery group was also included (control and high dose animals). No mortality, clinical signs were indicated. No effects on body weights were observed. Changes in liver weights were seen in treated males and females in all treatment groups. In addition changes in hematology were seen in all males at 50 mg/kgbw and higher and in females at 200 mg/kg bw and day. Mild changes in liver histology were observed in males and females of the mid and high dose groups. Histopathological changes in testis were noted in males treated with the test substance. Sperm rention was noted in males of the mid (6/6 mild) and high dose group (6/6 moderate) and in high dose males of the recovery group (5/6 moderate). Degeneration of step 19 spermatids were seen in males of the low (3/6 mild), mid (6/6 mild) and high (6/6 moderate) dose group and males of the high dose recovery group (5/6 moderate). Giant cell formation was seen in high dose males of the recovery group (4/6 moderate to market). In these males nuclear vacuolation of spermatids were found (4/6 mild to moderate). Moreover in the high dose males of the recovery group a decrease in germ cells were indicated (2/6 mild to marked). Whereas the effects seen in liver and hematology tend to be reversible, the adverse effects seen in the testis seems to be permanent or even more severe. Based on the findings of this study a LOAEL of 50 mg/kg bw and day is suggested for reproduction toxicity.

Adverse effects on testis were also noted in a subchronic toxicity study and a chronic feeding study in Wistar rats (Takagi 1994). In the subchronic feeding study the rats were fed with 0, 1200, 6000, 30000 ppm in the diet (for more details see chapter repeated dose toxicity). In males histological changes in the testis and a dose-dependent decrease in testis weights were noted in all treatment groups. At week 12 testicular atrophy was pronounced and was observed in all treated males. Decrease of spermatogenesis was evident in all treated males at week 4 and thereafter. Interstitial edema in the testis was also apparent in all treated rats at week 12. At week 4 and 12, epididymis atrophy and hypospermia was observed in high dose males and atrophy of the seminal vesicles and prostate in mid and high dose males. In females atrophy of the ovaries and uterus were apparent in mid and high dose females. Based on the findings of this study a LOAEL of 1200 ppm, according to 88 mg/kg bw/d is suggested for males, which based on the adverse effects on testis and spermatogenesis. For females a NOAEL of 1200 ppm, according to 104 mg/kg bw and day is suggested, which based on the effects on ovaries and uterus noted at 618 mg/kg bw and day.

In the chronic study Wistar rats were feed for 18 months with the test substance (for more details see chapter repeated dose toxicity). In males a decrease of the absolute and relative testes weights were observed at 1000 ppm. Several histopathological changes of the testis were noted in treated males. Testis tubules atrophy was observed (control 2/19, low: 4/19, mid: 1/18, high 19/19). Spermatogenic arrest were seen in male rats (control 2/19, low 1/19, mid: 1/8, high 19/19). In addition epididymis hypospermia was observed (control 2/19, low: 1/19, mid: 1/18, high: 19/19). The severity ranged from marked in the control males to severe in the treated animals. In females no significant changes were noted in ovaries. Based on the changes seen in male reproduction organs a NOAEL of 12.7 mg/kg bw/day for toxicity to reproduction is suggested for males and a NOAEL of 54.2 for females.

In a subchronic feeding study with Wistar rats (for more details see chapter repeated dose toxicity) adverse effects on reproduction organs were noted in treated animals (BAYER AG 1982). Males administered 1000 ppm and 3000 ppm (ca. 76 and 282 mg/kg bw/day) showed a significant reduction of testes weights and a severe atrophy of the testes. In addition, at the highest dose group (3000 ppm) a reduction of the epididymis was found. In females an atrophy of both uterus horns were seen at 3000 ppm (4/10). Based on the findings of this study a NOAEL for toxicity to reproduction of 24.91 for males and 113.16 for females is suggested.

In conclusion:

The data evaluation revealed that the test substance 6,6’-di-tert-butyl-2,2’-methylendi-p-cresol (DBMC) caused adverse effects on male reproductive organs and on fertility in rats (see discussion above).

A slight decrease in fertility was noted in a Reproduction/Developmental Toxicity Screening Test (OECD TG 421, MHWJ 1999) shown by a slight decrease of the number of pups born. This observation was accompanied by effects on the sperm quality and was related to a decrease in the number of corpora lutea and number of implantation scars (see discussion above).

Adverse effects like atrophy of the testis and epididymides, decrease in the total sperm number etc. were noted in male rats in a Reproduction/Developmental Toxicity Screening Test (OECD TG 421, MHWJ 1999). The adverse effects on the testis were confirmed by the findings of the repeated dose studies performed independently in different laboratories (see discussion above). As discussed and confirmed by recent literature (Mangelsdorf et al. 2003, Ulbrich & Palmer 1995, Janer et al. 2007, Dent 2007, Sanuissho et al. 2008) histopathological examination of reproductive tissues in repeated dose toxicity studies in rodents is of high value and sensitivity for evaluation of reproductive toxicity in males and females. Histopathological changes on the reproductive organs in repeated dose toxicity studies are indicative of effects on fertility. With this respect repeated dose toxicity studies should be considered sensitive and reliable information to evaluate toxicity on fertility if histological examination of the reproductive organs is covered.

Based on the above discussed findings on fertility and reproductive organs demonstrated in a Reproduction/ Developmental Toxicity Screening Test (OECD TG 421) and in repeated dose toxicity studies it is likely that the test substance in rats is toxic to reproduction at doses that lead to some but not overt toxicity. A generation reproductive toxicity study is not available.

According to regulation EC No 1907/2006 (REACH regulation) Annex IX, chapter 8.7.3. an Extended One-Generation Reproductive Toxicity study is required, if the 28-day or 90-day study indicate adverse effects on reproductive organs or tissue. Based on the recommendation given in regulation EC No. 1907/2006 and the findings obtained in the Reproduction/Developmental Toxicity Screening Test (MHWJ 1999) and the repeated dose toxicity studies (discussed above) an Extended One-Generation Reproductive Toxicity study is proposed to be conducted and provisionally the test substance is classified in category 2 according to regulation (EG) 1272/2008 CLP). Final classification would be discussed depending on effects observed in the EOGRTS.


Short description of key information:
The data evaluation revealed that the test substance 6,6’-di-tert-butyl-2,2’-methylendi-p-cresol (DBMC) caused adverse effects on male reproductive organs and on fertility in rats.
A slight decrease in fertility was noted in a Reproduction/Developmental Toxicity Screening Test (OECD TG 421) (MHWJ 1999) shown by a slight decrease of the number of pups born. This observation was accompanied by effects on the sperm quality and was related to a decrease in the number of corpora lutea and number of implantation scars. Adverse effects like atrophy of the testis and epididymides, decrease in the total sperm number etc. were noted in male rats in a Reproduction/Developmental Toxicity Screening Test (MHWJ 1999). The adverse effects on the testis were confirmed by the findings of the repeated dose studies performed independently in different laboratories. Based on the findings of the Reproduction/Developmental Toxicity Screening Test and the findings from the repeated dose toxicity studies it is likely that the test substance in rats is toxic to reproduction at doses that lead to some but not overt toxicity.

Based on the recommendation given by regulation EC No 1907/2006 an Extended One-Generation Reproduction Toxicity study is proposed to be conducted and provisionally the test substance is classified in category 2 according to regulation (EG) 1272/2008 CLP). Final classification would be discussed depending on effects observed in the EOGRTS.

Effects on developmental toxicity

Description of key information

There is no evidence of a specific developmental toxicity based on a reproductive screening assay and a developmental toxicity study. The NOAEL for maternal toxicity was assessed to be 50 mg/kg bw/ day and the NOAEL for pup development is considered to be 50 mg/kg bw/day based on the results from the OECD Guideline study 421(MHWJ 1999). Based on the developmental toxicity study (Tanaka 1990) the NOAELs were considered to be 93.5 mg/kg bw/day for maternal toxicity and 187 mg/kg bw/day for foetal toxicity.
Based on the data from the TG 421 a NOAEL developmental toxcity of 50 mg/kg bw and day is suggested.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
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)
Qualifier:
according to
Guideline:
other: OECD Guideline 421 (Reproduction/Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Crj: CD(SD)
Details on test animals and environmental conditions:
Weight at study initiation: (P) Males: 332-383 g; Females: 206-238 g
Route of administration:
oral: gavage
Vehicle:
other: in 5% gum arabic
Details on mating procedure:
Male/female per cage: 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
Duration of test:
male: 50-52 d, female: 40-48 d (from 14 days before mating to the day 3 of lactation)
No. of animals per sex per dose:
12/per dose group/sex
Control animals:
yes, concurrent vehicle
Details on study design:
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
Maternal examinations:
BODY WEIGHT: Yes,
DETAILED CLINICAL OBSERVATIONS: Yes
FOOD CONSUMPTION: Yes
POST-MORTEM EXAMINATIONS: Yes


Ovaries and uterine content:
lengh of gestation, corporal lutea, implantation scars, implantation index, gestation index, pups born, delivery index, live pups born, sex ratio at birth. birth index,
Fetal examinations:
Dead pups on day 0 of lactation, live birth index, live pups on day 4 of lactation, viability index, external anomalies, body weights of pups
Statistics:
Dunnett's or Scheffe's test for continuous data and Chi square test for quantal data
Indices:
live birth index, viability index, implantation index, gestation index, delivery index
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
decrease in body weight gain,
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
NOAEL
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 (200 mg/kg bw): Pup numbers on day 0 and 4 of lactation were decreased
Abnormalities:
not specified
Developmental effects observed:
not specified

Dams:

Females (200 mg/kg bw):Suppression 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

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

Organ weight of female rats

 Dose (mg/kg) 12.5  50  200  800 
 number of dams 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**
 Ovaries (mg)  94.50 ± 12.06  91.43 ± 10.00   89.88 ± 8.77   89.69 ± 16.74   88.78 ± 14.65

significantly different from control (**p<0.01)

Number of estrous cases and reproductive performance

 Dose (mg/kg) 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

Observation of pups

Dose (mg/kg) 12.5  50  200  800 
 Number of dams  12 12  12  12  12 
 Lengh of gestation (days)  22.08 ± 0.29  22.00 ± 0.00 22.33  ± 0.49  22.45 ± 0.52   22.27 ± 0.65
 Corpa 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*
 Implantation index  86.9 ± 14.7  91.0 ± 12.8   93.6 ± 7.4  90.1 ± 8.5   93.1 ± 7.2
 Gestation index (%)  100 100  100  100  83.3 
 Pups born  13.5 ± 3.3  13.5 ± 1.0    14.8 ± 1.3  11.7 ± 1.4**   12.2 ± 1.8*
 Live pups born   13.1 ± 3.2  13.3 ± 0.8   14.3 ± 1.3  11.5 ± 1.0**   11.3 ± 4.1
 Birth index (%)  90.7  ± 9.4 91.2  ± 4.8  94.7 ±  4.4   86.0 ± 9.6   83.8 ± 28.8
 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 ± 30.0
 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
 Viability index (%)  100.0  ± 0.0 100.0 ± 0.0  99.4 ± 2.0  98.5 ± 3.4  100.0 ± 0.0
 External anomalies (%)  0.0 ± 0.0   0.0 ± 0.0  0.0 ± 0.0   0.0 ± 0.0   0.0 ± 0.0 
 Body weight of pups          
 Male Day 0  6.78 ± 0.40   6.81 ± 0.23  6.90 ± 0.50  7.60 ± 0.52**  6.97 ± 0.71
 Male Day 4   11.02 ± 0.83  11.05 ± 0.77  10.58 ± 1.11  11.29 ± 1.14  9.68 ± 1.72*
 Female Day 0   6.43 ± 0.37  6.40 ± 0.17  6.53 ± 0.44  7.25 ± 0.49**  6.61 ± 0.66
 Female Day 4   10.33 ± 0.81  10.36 ± 0.67   10.20 ± 1.15  10.77 ± 1.04   9.30 ± 1.64

Significantly different from control (*p<0.05, **p<0.01)

Executive summary:

In a reproduction/developmental toxicity screening test (OECD Guideline 421) with rats toxic effects of the test substance Vulcanox BKF were seen. In dams 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. No external anomalies were found in any of the pups evaluated

The maternal NOAEL for developmental toxicity was assessed to be 50 mg/kg bw/ day and the NOAEL for pup development is considered to be 50 mg/kg bw/day (MHWJ 1999).

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

In the reproduction/developmental toxicity screening test (OECD TG 421) with Sprague-Dawley rats (described above) toxic effects of the test substance 6,6’-di-tert-butyl-2,2’-methylendi-p-cresol (DBMC) were noted in treated dams and their pups. In these dams a suppression of the body weight gain was found 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. The number of live pups born and live pups on day 4 of lactation was slightly decreased compared to the corresponding control. Moreover, pups of the 800 mg/kg bw/day dose group showed a decrease in body weights compared to the control. However, no external anomalies were found in any of the pups evaluated. The authors concluded that the maternal and foetal NOAEL for developmental toxicity is 50 mg/kg bw/ day (MHWJ 1999).

In a developmental toxicity study with Wistar rats maternal toxic effects of the test substance 6,6’-di-tert-butyl-2,2’-methylendi-p-cresol were observed. The maternal toxic effects determined were suppression of body weight gain, decrease in food consumption and toxic signs such as diarrhoea at concentrations of 187 and 375 mg/kg bw and day. In addition, foetal toxicity was seen at 375 mg/kg bw and day indicated by an increase in foetal death. No teratogenic effects were found in external, visceral and skeletal observations (Tanaka 1990). The NOAELs were considered to be 93.5 mg/kg for maternal toxicity and 187 mg/kg/day for foetal development.

Justification for classification or non-classification

Based on the findings from the Reproduction/Developmental Toxicity Screening Test and the repeated dose toxicity studies provisionally the test substance is classified in category 2 according to regulation (EG) 1272/2008 CLP).