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Toxicological information

Repeated dose toxicity: oral

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Administrative data

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)
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
publication
Title:
Acute, subchronic and chronic toxicity studies of a synthetic antioxidant, 2,2'-methylenebis(4-methyl-6-tert-butylphenol) in rats
Author:
Takagi, A.; et al.
Year:
1994
Bibliographic source:
The Journal of Toxicological Sciences, vol. 19, 77-89

Materials and methods

Principles of method if other than guideline:
other: chronic toxicity feeding study
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
2,2'-methylenebis(6-tert-butyl-4-methylphenol), test substance from Ouchi Shinko Chemical Ind. Ltd.

Test animals

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)

Administration / exposure

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
Doses / concentrations
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

Examinations

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

Results and discussion

Effect levels

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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

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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).