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Administrative data

Description of key information

Several repeated dose toxicity studies are available for the test substance 6,6'-di-tert-butyl-2,2'-methylenedi-p-cresol. Comparable effects were seen in subacute, subchronic and chronic toxicity rodent studies. Effects on body weight gain, increases in liver weights and adverse effect on reproduction organs were noted (effects on reproduction organs see discussion chapter toxicity to reproduction). In the subchronic feeding study with dogs effects on the liver were noted at 330 ppm (ca. 11 mg/kg bw/day). However, the number of dogs used is very limited and thus the study is used only for supporting reasons. Based on the findings from the several rodent toxicity studies a NOAEL of 12.7 mg/kg bw and day is used for DNEL calculation, which based on a suppression of body weight gain, increase in liver weight, decrease in testis weight, and histopathological lesions in the testis and the epididymis observed at 42.3 mg/kg bw/ day in the chronic feeding study with Wistar rats (Takagi 1994).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable, well-documented publication which meets basic scientific principles (limitations: no data to test substance homogeneity and stability given, small number of animals evaluated at study termination)
Reason / purpose:
reference to same study
Principles of method if other than guideline:
other: chronic toxicity feeding study
GLP compliance:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
the animals was maintained at tepmerature of 24 +/- 1°C and 55 +/- 5% humidity with a 12 h light/dark cycle; rats were housed in plastic cages (5 rats/cage)
Route of administration:
oral: feed
Vehicle:
other: test substance contained in the diet
Details on oral exposure:
pellets of diet containing the substance; stored at 4°C until use
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
18 months
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
30 per dose and sex
Control animals:
yes, plain diet
Details on study design:
Age at study initiation: 5 weeks old for both sexes; weight at study initiation: 393 +/- 21 g male, 230 +/- 15 g for female; no. of animals per sex and per dose: 30 (5 animals/group were sacrified after 6 and 12 months for hematological serum biochemical examinations)
Positive control:
no data
Observations and examinations performed and frequency:
Clinical observations performed and frequency: general conditions was observed daily; body weight and food consumption were determined monthly; hematological and serum biochemical examination were performed for 5 animals/sex/dose group at 6, 12, 18 months

Organs examined at necropsy:
Organ weight: brain, heart, lungs, liver, kidney, spleen, adrenals, testes, ovaries, pituitary and thyroid glands

Microscopic evaluation /all groups: brain, heart, lungs, liver, kidney, spleen, adrenals, testes, ovaries, pituitary and thyroid glands, salivary glands, esophagus, stomach, small and large intestine, pancreas, urinary bladder, seminal vesicles, epididymis, ischiac nerve, uterus, prostate, mesenteric lymph nodes, thymus, spinal cord, skeletal muscle, bone marrow
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
Analysis of variance (ANOVA) techniques with Dunnett's or Scheffe's test for continous data and Chi square test for analysis of categorical data
Dose descriptor:
NOAEL
Effect level:
12.7 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: slight increase in relative liver weights
Dose descriptor:
NOAEL
Effect level:
15.1 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effects
Dose descriptor:
LOAEL
Effect level:
42.3 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: decreased body weights, increased absolute and relative liver weights, decreased absolute and rel. testis weights, atrophy of testicular tubules and spermatogenic arrest and epididymis, hypospermia
Dose descriptor:
LOAEL
Effect level:
54.2 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: decreased body weights, increased absolute and relative liver weights
Critical effects observed:
not specified

NOAEL Males: 300 ppm (12.7 mg/kg bw/ day), females 300 ppm (15.1 mg/kg bw/day)

LOAEL Male: 1000 ppm (42.3 mg/kg/day), Female: 1000 ppm (54.2 mg/kg/day)

Clinical signs: no remarkalbe changes in general appearance were observed in any rat. survival rates in all treated animals were comparable to those of control (see Table below).

Body weight: Significant suppression of body weight gain was observed from the month 6 in the male group at 1000 ppm and from the month 1 in the female group at 1000 ppm.

Food/water consumption: No significant effect was observed during the study.

Table: survival rate, body weight and food consumption: effects seen after 18 months

 

 Males         

 Females         

 Diet level (ppm)  0 100  300  1000   0  100  300  1000

Final mean body weight (g)

 545  528  520  498*  375  368 353   278*
 Mean food intake (g/rat/day)  16.6  16.7  16.6  16.2  12.2  12.7  12.2  11.5
 Survival rate (%)  95  95  91  95  90  100  95  95

(* significant: not specified the degree)

Haematology: In the hematological and serum biochemical analysis, several parameters demonstrated significant alternation. However, none appeared to be of biological significance, since they did not show the same tendency throughout the experimental period and/or the degrees of change were very small.

Mortality and time to death: Survival rates in all treated groups were comparable to those of control.

Gross pathology incidence and severity: Organ weight changes:

Male: Increase in liver weight at 300 ppm (absolute (p<0.05) and relative (p<0.01)); decrease in testis weight at 1000 ppm (absolute and relative) (p<0.01)

Female: Increase in liver weight at 1000 ppm (relative) (p<0.01). no changes in ovary weights were observed in any of the treated females. no changes in other organs were observed in any treated groups for males and females.

Table: Organ weightseffects seen after 18 months

 

 Males         

  Females        

  Diet level (ppm)  0  300  1000   0  1000
 Absolute weight          
 Liver (g, Mean ± SD)  12.28 ± 0.93  12.59  14.19 ± 1.35*   7.60 ± 0.88   7.39 ± 0.83
  Testis (g, Mean ± SD)  3.28 ± 0.48  3.98  ± 0.54  0.82 ± 10.18**    
  Relative weight          
 Liver (g%, Mean ± SD)   2.37 ± 0.16  2.58 ± 0.16**#  3.00 ± 0.13**   2.08 ± 0.15   2.79 ± 0.35**
  Testis (g%, Mean ± SD)   0.63 ± 0.10  0.82 ± 0.11  0.17 ± 0.05*    

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

# relative liver weight 8.9 % increased compared to control

Histopathology (incidence and severity):

Histopathological lesions were only observed in the testis and epididymis of males. However, no interstitial cell tumors were apparent. In the other organs of males, no changes induced by the test substance were observed and no changes in any organs were apparent in females. No neoplastic lesions which could be attributed to the test substance were observed in any organs of males and females.

Male: Atrophy of testicular tubules and spermatogenic arrest and epididymis

hypospermia were observed in the 1000 ppm group.

Female: No significant effect was observed.

Table: Histopathological effects seen after 18 months

 Diet level (ppm)  degree*  0  100  300  1000
 No. of animals    19  19  18 19 

 Testis, tubules Atrophy

  ±  0  0  0
   +  0  0  0  0
   ++  2  0  0  0
   +++  0  3  1  19
 Spermatogenic arrest  ++  2  0  0  0
   +++  0 19 
 Epididymis Hypospermia  ++  2  0  0
   +++  0 19 

CONCLUSIONS: Toxic effects in this study are suppression of body weight gain, increase in liver weight, decrease in testis weight, and histopathological lesions in the testis and the epididymis.

The NOAELs are 12.7 mg/kg/day (300 ppm) for male and 15.1 mg/kg/day (300 ppm) for female.

Executive summary:

In a chronic feeding study male and female Wistar rats were administered with 0, 100, 300 and 1000 ppm test substance in the diet. A suppression of the body weight gain was noted at 1000 ppm (corresponding to 42.3 mg/kg bw/d for males and 54.2 mg/kg bw/d for females). An increase in the relative liver weight was found at 300 ppm and 1000 ppm for males and 1000 ppm for females. In males a decrease of the absolute and relative testes weights and atrophy of testicular tubules were observed at 1000 ppm. In addition a spermatogenic arrest and epididymis hypospermia were noted at this concentration. The NOAELs are considered to be 12.7 mg/kg/day (300 ppm) for male and 15.1 mg/kg/day (300 ppm) for female (Takagi 1994).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
12.7 mg/kg bw/day
Study duration:
chronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated dose toxicity: oral

In a subchronic feeding study male and female Wistar rats (10 rats per dose and sex) were feed with the test substance 6,6'-di-tert-butyl-2,2'-methylenedi-p-cresol (DBMC) in the diet for 13 weeks (Bayer AG, 1982). The doses  applied were 100, 330, 1000 and 3000 ppm, according to  about 7.4, 25, 75, and 280 mg/kg bw/d for males and 10, 30, 113,  and 345 mg/kg bw/d for females. No mortality or clinical signs were noted during the study. No biological relevant changes were noted in clinical chemistry. In male and female rats a significant decrease in the body weight gain and an increase in liver weights were noted at the highest dose groups (1000 ppm and 3000 ppm). Males administered 1000 ppm and 3000 ppm 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 female rats a reduction of the uterus and an atrophy of both horns of the uterus were seen at 3000 ppm. The authors concluded that the NOAEL is 330 ppm (males: 24.91 and females: 31.3 mg/kg bw/day) (Bayer AG, 1982).

These findings were supported by further repeated dose studies with Wistar rats.

In a subchronic feeding study with Wistar rats toxic effects of the test substance DBMC were noted (Takagi 1994). The rats were fed for 12 weeks with 0, 1200, 6000, 30000 ppm in the diet (corresponding to 0, 88, 564, 3120 mg/kg bw/ day for males and 0, 104, 618, 2610 mg/kg bw/d for females). Mortality occurred in males of the mid and high dose groups (survival rate 45 % and 20 %) and in females of the high dose group (survival rate 36%). Mortality was accompanied by haemorrhage from nasal cavity. Diarrhoea was observed in four males of the high dose group and one female of the high dose group. Several biochemical changes were observed in males and females of the mid and high dose group. No changes in biochemical urine parameter were revealed. A significant decrease in red blood cells was observed in mid dose males at week 12. Moreover, a significant decrease of haemoglobin content was observed in high dose males at week 4, and mid dose males at week 12 and all treated females. Decreased food intake was observed in males of the mid and high dose groups and in females of the high dose group. The body weight gain was reduced and the relative liver weights were increased at 6000 ppm and higher in males and the relative and absolute liver weights were increased at 6000 ppm and above in females. In both sexes, thymus atrophy and bone marrow hypoplasia were noted at 6000 and 30000 ppm. In males histological changes in the testis and a dose-dependent decrease in testis weights were noted in all treatment groups (for more details see chapter toxicity to reproduction). Thus, no NOAEL could be established for males. In females histopathological changes in ovaries and uterus and a decrease in ovary weights were noted at a concentration of 6000 ppm and higher (Takagi 1994). The NOAEL was 1200 ppm (104 mg/kg bw) in female rats.

In a chronic feeding study from the same group (Takagi, 1994) male and female Wistar rats were administered with 0, 100, 300 and 1000 ppm test substance in the diet for 18 months. The mid and high dose correspond to 12.7 and 42.3 mg/kg bw/d for males and 15.1 and 54.2 mg/kg bw/d for females, respectively. No data is given for the low dose. No signs of toxicity were indicated related to test substance treatment. In addition, survival of treated rats was comparable to that of control animals. A suppression of the body weight gain was noted at 1000 ppm. An increase in the relative liver weight was found at 300 ppm and 1000 ppm for males and 1000 ppm for females. In males a decrease of the absolute and relative testes weights and atrophy of testicular tubules were observed at 1000 ppm. In addition a spermatogenic arrest and epididymis hypospermia were noted at this concentration. In the hematological and serum biochemical analysis, no biological relevant changes were noted. The NOAELs are considered to be 12.7 mg/kg/day (300 ppm) for males and 15.1 mg/kg/day (300 ppm) for females (Takagi 1994).

In two repeated dose studies with Sprague-Dawley rats similar toxic effects were found.

In a reproduction toxicity screening study (OECD TG 421) the oral toxicity of the test substance was evaluated at doses of 0, 12.5, 50, 200, and 800 mg/kg bw/day (for more details see chapter toxicity to reproduction). Toxic effects in this study found were a suppression of body weight gain, low food consumption, and histopathological lesions in the testis and the epididymis (for more details see chapter toxicity to reproduction). The NOAELs for systemic toxicity are considered to be 12.5 mg/kg/day for males and 50 mg/kg/day for females (MHWJ 1999).

In a subacute toxcity study with Sprague-Dawley rats males and females were administered per gavage with 0, 50, 200, 800 mg/kg bw/day test substance (six animals per dose group and sex). A14-day recovery group, consisting of control and high dose animals (6 per dose and sex), was also included. No mortality or clinical signs were reported. No relevant changes in body weights were noted in any of the treated animals compared to the corresponding controls. An increase in liver weights was seen in all treatment groups in both sexes. In addition mild centrilobular hepatocyte hypertrophyh was seen in males and females at 200 mg/kg bw/day (1/sex) and above (1m/2f). Lesions of the testis were noted at 200 mg/kg bw/day and higher, and sperm abnormality at 50 mg/kg bw/day and higher in treated males. In females an increase in adrenal weights was noted at 200 mg/kg bw/day and higher. Furthermore, a prolongation in PT and APTT were seen in males in all treatment groups and in females at 200 mg/kg bw/day and higher. The authors considered that the NOAEL is less than 50 mg/kg bw/day for males and females (MHWJ 1996).

The test substance 6,6'-di-tert-butyl-2,2'-methylenedi-p-cresol was also evaluated in a subchronic toxicity study in dogs (American Cyanamid Company 1965). Diets containing the test substance were fed to groups of purebred beagles, 2 males and 2 females per group. Two separate trials were conducted. In the first, animals 5 to 6 months of age were fed diets containing 0 ppm, 330 ppm, 1000 ppm or 3000 ppm test substance for 135 to 142 days. In the second trial animals were 9 to 12 months of age. They were offered a control diet or diet containing 100 ppm or 200 ppm of test substance for 122 to 123 days. Mortality occurred in the first trial at 3000 ppm.; one male of the high dose group died after 59 days treatment and one high dose female after 113 days treatment. No test substance related clinical signs were noted in any of the treated animals in both trials. Food intake and body weight gain was not affected up to 1000 ppm, whereas the surviving 3000 ppm male had a higher weight gain compared to the lower dose groups and the 3000 ppm female clear reduced body weight gain compared to the lower dose groups. There were no significant differences in mean haemoglobin, hematocrit or total leukocyte counts between control and test animals. No adverse effect on plasma prothrombin activity was noted in any of the treated animals. An increase in plasma alkaline phosphate activity was noted at 200 ppm and higher. The organ weights noted for liver, kidney, spleen, pancreas, adrenals, brain, heart and pituitary were within the normal control data range. The surviving 3000 ppm male had a relative high thyroid weight and one 100 ppm male had a relative low testes weight. The relevance of these findings was discussed as questionable. Histological changes of the liver were seen at 330 ppm (ca. 11 mg/kg bw/day) and higher (e.g. inflammatory and hyperplastic changes consisting of diffuse mononuclear hepatitis, bile duct hyperplasia, dissociation and disarray of hepatic cord cells, and focal tiny hemosiderin granulomas). Animals feed 100 and 200 ppm (ca. 7 mg/kg bw/day) had no such lesions. All other organs evaluated (e.g. stomach, ileum, colon, urinary bladder, gonads, prostate or uterus, adrenal, kidney, thyroid (with parathyroid where included), lung, heart, pituitary and brain) did not have significant lesions or changes of any great incidence.


Repeated dose toxicity: via oral route - systemic effects (target organ) urogenital: testes

Justification for classification or non-classification

Classification is not required based on the classification criteria 67/548/EWG and regulation no. 1272/2008 (GHS).