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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Conducted according to sound scientific principles, similar to OECD TG 421. GLP.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1998

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Tris(2-ethylhexyl)benzene-1,2,4-tricarboxylate
Source: Daihachi Kagaku Kogyo Co., Ltd. Lot. No. N-80301
Purity: >99.0% Kept at room temperature in a dark place until use.

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
Age at study initiation: 10 week old for both sexes.
Weight at study initiation: 373-435 g for males, 217-257 g for females
No. of animals per sex per dose: 12 per sex per dose group

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Administration period: Males for 46 days from 2 weeks prior to mating; Females from 2 weeks prior to mating to day 3 of lactation
Post exposure observation period: None
Terminal kill: Males day 47; Females day 4 of lactation
Details on mating procedure:
Male/female per cage; 1/1, length of cohabitation; with in the limit of 14 days until proof of pregnancy (formation sperm detection in vagina) was observed.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Males for 46 days from 2 weeks prior to mating; Females from 2 weeks prior to mating to day 3 of lactation
Frequency of treatment:
once a day
Doses / concentrations
Remarks:
Doses / Concentrations:
0(vehicle), 100, 300, 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
12 per sex per dose group
Control animals:
yes, concurrent vehicle
Details on study design:
Administration period: Males for 46 days from 2 weeks prior to mating;
Females from 2 weeks prior to mating to day 3 of lactation
Post exposure observation period: None
Terminal kill: Males day 47; Females day 4 of lactation

The animals were sacrificed on the day 4 of lactation for females.
Males and females with no mated were killed 1 day after the mating period.
Females with no delivery killed 26th day of gestation period.
Vehicle: Corn oil
Mating procedures: Male/female per cage; 1/1, length of cohabitation; with in the limit of 14 days until proof of pregnancy (formation sperm detection in vagina) was observed.

Examinations

Parental animals: Observations and examinations:
Clinical observations performed and frequency:
Parent: General appearance once a day

Parameters assessed during study:
Body weight.
Males: Prior to the first dosing and 2, 5, 7, 10, 14 day. After that once a week, the day sacrificed.
Females: Prior to the first dosing and 2, 5, 7, 10, 14 day. During gestation period, 0, 1, 3, 5, 7, 10, 17 and 20 day.
During lactation period, 0, 1, and 4.
During cohabitation period, the same day with male.

Food/water consumption.
The same day when body wt. determined, except lactation period and the day sacrificed for males, also, 0 day of gestationand lactation for female.
Sperm parameters (parental animals):
Microscopic:
Males: Testis and epididymis. Count of sertoli sells, spermatocytes, round spermatids and elongate spermatids in seminiferous tubules of 5 animals of all dosing groups.(Stage I-VI, VII-VIII, IX-XI, XII-XIV of spermatozoon formative cycle.)
Litter observations:
Clinical observations performed and frequency:
Pups: General appearance once a day after birth

Parameters assessed during study:
Body weight.
Pups: Day 0 and 4
Postmortem examinations (parental animals):
Organs examined at necropsy:
Parent: Males and females: Gross pathlogy of all organs were tested.
Males: Organ weight: Testis and epididymis of all animals.
Female: Organ weight: Ovary of all animals.

Count: Implantation sites and corpus luteum of ovary of all animals.

Microscopic:
Males: Testis and epididymis. Count of sertoli sells, spermatocytes, round spermatids and elongate spermatids in seminiferous tubules of 5 animals of all dosing groups.(Stage I-VI, VII-VIII, IX-XI, XII-XIV of spermatozoon formative cycle.)
Females: Ovary

Postmortem examinations (offspring):
Pup: Gross pathlogy of all organs were tested. Dead pups and abnormal organs were tested histopathogy.
Statistics:
Chi square test for 1 grade positive data and Fisher's test for another. Bartlett's test or Kruskal-Wallis' test for 2 or more grade positive data. And used Dunnett's test or Mann-Whitney's U-test for examination.
Reproductive indices:
No. of pairs with successful copulation;
Copulation index (No. of pairs with successful copulation/No. of pairs mated) x 100, duration of mating;
No. of pregnant females;
Fertility index = (No. of pregnant animals/No. of pairs with successful copulation) x 100
Offspring viability indices:
No. of corpora lutea;
No. of implantation sites;
Implantation index (No. of implantation sites/No. of corpora lutea) x 100;
No. of pups born, delivery index (No. of pups born/No. of implantation sates) x 100;
No. of live pups born;
Live birth index (No. of live pups born/No. of pups born) x 100, sex ratio of pups;
No. of dead pups born;
Gestation length;
Gestation index (No. of females with live pops delivered/ No. of pregnant females) x 100;
Nursing index (No. of females nursing live pups/No. of females with normal delivery) x 100;
No. of live pups on day 4, viability index (No. of live pups on day 4/No. of live pups born) x 100.

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No adverse effects were observed that affected reproductive function
Reproductive performance:
no effects observed

Details on results (P0)

Mortality and day of death: None

Body weight: No statistical significant difference from controls.

Food/water consumption: No statistical significant difference from controls.

Reproductive data: No statistical significant difference from controls.

Grossly visible abnormalities, external, soft tissue and skeletal abnormalities
Males:
For the control group, atrophy of seminiferous tubules were observed 2 animals and cell debris were observed. There was a slight decrease of spermatids observed in one of the control animals. In the experimental animals, a slight decrease of spermatocytes and spermatids was observed in 2 animals (300 mg/kg) and in 11 animals (1000 mg/kg). A moderate decrease of spermatocytes and spermatids was observed in one animal of 1000 mg/kg/dosing group where a few multinucleate giant cell were noted and a slight vacuolization of sertoli sells were observed. This single animal also had a moderate amount of cell debris, a moderate decrease of spermatids, and a slight granuloma of spermatic in the epididymis.

Number of cells in seminiferous tubules:
Group 1(Stage I-VI): A statistically significant decrease in the number of spermatids at the 300 mg/kg dosing group were observed; a statistically significant decrease in the number of spermatocytes and spermatids were observed for the 1000 mg/kg dosing group.
Group 2(Stage VII-VIII): A statistically significant decrease in the number of round spermatids and ratio of sertoli cells at 1000 mg/kg.
Group 3(stage IX-XI): A statistically significant decrease in the number of elongate spermatids and ratio of sertoli cells at 1000 mg/kg.
Group 4(stage XII-XIV): A statistically significant decrease in the number of spermatocytes, elongate spermatids, and ratio of sertoli cells at 1000 mg/kg dosing group.

Females:
Cyst of corpus luteum of ovary was observed 2 animals of 300 mg/kg dosing group. No abnormal ovaries were observed in the control females 100 mg/kg dosing without successful copulation, or in the 100 mg/kg dosing group without pregnant.

There were no effects observed in any of the following parameters:
1) No. of pairs with successful copulation;
2) Duration of mating ;
3) Copulation index(%);
4) No. of pregnant animals,
5) Fertility index(%);
6) No. of corpora lutea;
7) No. of implantation sites;
8) Implantation index(%);
9) No. of pups born(%);
10) Delivery index(%);
11) Live pups born;
12) Live birth index(%);
13) Sex ratio(M/F);
14) Dead pups born;
15) Gestation length(day);
16) Gestation index(%);
17) Nursing index(%)

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no adverse effects observed to fertility for male or for female rats.
Dose descriptor:
NOEL
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No effects were observed at the highest dose tested.
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

Body weight and weight gain of 300 mg/kg dosing group for both sexes were slightly low. But all pups of 100 and 1000 mg/kg dosing group were not statistical significant difference from controls. At the other tests, no statistical significant difference from controls.

1) No. of pups born (%) – no effects observed;
2) Delivery index (%) – no effects observed;
3) Live pups born – no effects observed;
4) Live birth index(%) – no effects observed;
5) Sex ratio (M/F) – no effects observed;
6) Dead pups born – no effects observed;
7) Live pups on day 4 – no effects observed;
8) Viability index (%)– no effects observed;
9) Body weight of pups – only the 300 mg/kg dosing group for both sexes were statistically significantly decreased;
10) Day 0-4, weight-gain (M and F, in g) – only the 300 mg/kg dosing group for both sexes were statistically significantly decreased;
11) Body weight gain (%) (M and F) – no effects observed.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no adverse developmental effects noted for the F1 pups.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1 - Histopathological finding in rats

  Dose (mg/kg bw/day)
Histopathological endpoint 0 100 300 1000
# of male rats examined 12 12 12 12
         
Testis:        
Decrease, spermatocyte and spermatid        
Total 0 0 2 12
Slight 0 0 2 11
Moderate 0 0 0 1
         
Multinuclear giant cell, seminiferous tubule (slight) 0 0 0 1
Vacuolozation, Sertoli cell (slight) 0 0 0 1
Atrophy, seminiferous tubule (slight) 2 0 0 0
Epididymis:        
Cell debris, lumen        
Total 2 0 0 1
Slight 2 0 0 0
Moderate 0 0 0 1
Decrease, sperm        
Total 1 0 0 1
Slight 1 0 0 0
Moderate 0 0 0 1
Granuloma, spermatic (slight) 0 0 0 1
         
#. of female rats examined 12 12 12 12
Ovary:        
Cyst, corpus luteum 0 0 2 0

Table 2 - Number of cells in seminiferous tubules of male rats.

  Dose (mg/kg bw/day)
Histopathological endpoint 0 100 300 1000
# of male rats examined 5 5 5 5
         
Group 1 (Stage I-VI)        
No. of Sertoli cells (S.D.) 20.12(3.18)  19.08(1.49) 18.52(1.45) 18.08(1.45)
Spermatogonia No. 16.80(5.65) 20.52(2.58) 18.48(3.17)  15.76(2.61)
ratio a) 0.85(0.29) 1.08(0.19) 1.01(0.21) 0.87(0.11)
Spermatocytes No. 50.80(7.44) 51.80(4.84) 42.64(2.63)  40.84(5.63)*
ratio 2.53(0.13) 2.72(0.26) 2.37(0.24) 2.25(0.16)
Round spermatids No. 138.36(17.20) 128.00(8.89) 117.68(5.59)*  112.60(3.11)**
ratio 6.91(0.35)  6.75(0.84) 6.39(0.70)   6.26(0.48)
Elongate spermatids No. 130.00(21.71) 132.32(11.17) 103.28(12.34)* 95.36(8.44)**
ratio 6.53(1.15) 6.98(0.88) 5.62(0.90)  5.30(0.69)
         
Group 2 (Stage VII-VIII)        
No. of Sertoli cells (S.D.) 16.96(2.63) 17.04(2.17) 16.64(2.73) 16.52(2.23)
Spermatogonia No. 2.92(1.06)  2.40(0.93)  2.04(0.68)  2.60(1.10)
ratio a) 0.18(0.09)  0.14(0.05) 0.12(0.03) 0.16(0.06)
Spermatocytes No. 91.68(10.37)  94.68(6.55)  84.44(6.99)  82.32(6.70)
ratio 5.45 (0.56)  5.60(0.51)  5.16(0.79) 5.03(0.54)
Round spermatids No. 142.08(13.39) 131.64(13.72)  123.96(8.23)  118.76(8.28)*
ratio 8.45(0.62)   7.75(0.39) 7.66(1.66) 7.25(0.62)*
Elongate spermatids No. 129.24(17.37)  128.32(16.88) 114.72(9.80) 105.65(13.47)
ratio 7.78(1.54)  7.56(0.72)  7.09(1.62)   6.46(1.05)
         
Group 3 (Stage VII-VIII)        
No. of Sertoli cells (S.D.) 19.28(1.92)  20.52(1.55)  19.20(1.58) 19.32(2.18)
Spermatogonia No. 4.52(1.32) 4.20(1.50)  4.92(1.63)   3.32(1.02)
ratio a) 0.23(0.05) 0.21(0.08) 0.26(0.11)   0.18(0.05)
Spermatocytes No. 102.52(10.83)  99.08(8.42) 97.56(4.50)  89.04(9.00)
ratio 5.34(0.56)  4.85(0.50)  5.10(0.36)  4.62(0.32)
Elongate spermatids No. 145.24(11.01)  130.64(9.90))  131.68(19.71)  119.24(15.90*
ratio 7.56(0.61)  6.37(0.23) 6.88(1.04)  6.21(0.83)*
         
Group 4 (Stage VII-VIII)        
No. of Sertoli cells (S.D.) 19.16(2.81)  20.92(1.73) 18.64(1.72)  16.72(0.92)
Spermatogonia No. 4.04(0.89)  3.72(0.72)  3.64(0.48)  3.64(0.71)
ratio a) 0.21(0.05) 0.18(0.03) 0.20(0.02)  0.22(0.05)
Spermatocytes No. 109.80(13.15)  110.36(9.22)   99.44(4.54)   88.76(4.33)**
ratio 5.76 (0.29)  5.28(0.12)  5.36(0.34)   5.32(0.46)
Elongate spermatids No. 159.76(15.91)  150.28(18.99)  137.08(17.70)  105.16(18.34)**
ratio 8.39(0.63)  7.19(0.71)  7.35(0.62)  6.33(1.31)**
         
Values are expressed as Mean(S.D.)
 
a): (No. of spermatogenic cells/no. of sertoli cells in a seminiferous tubule)

Table 3 - Influence on reproductive performances of rats

  Dose (mg/kg bw/day)
Endpoint 0 100 300 1000
# of male rats examined 12 12 12 12
         
No. of male animals examined 12 12 12 12
No. of pairs with successful copulation 12 12 12 12
Duration of mating (day, Mean, (SD)) 2.1(1.2) 2.3(1.3) 2.7(1.2) 2.7(1.1)
Copulation index(%)* 100 91.7 100 100
No. of pregnant animals 11 10 12 12
Fertility index(%)** 91.7 90.9 100 100
         
*(No.of pairs with successful copulation/no.of pairs mated) x 10
**(No. of pregnant animals/no.of pairs with successful copulation) x 100
 

Table 4 - Influence on developmental performances of rats

  Dose (mg/kg bw/day)
Endpoint 0 100 300 1000
         
No. of corpora lutea 16.8(1.5) 17.3(1.3)  17.0(2.3)  17.9(2.2)
No. of implantation sites 15.5(1.7)  16.6(1.3) 16.0(2.0)  16.3(2.3)
Implantation index(%) a) 92.5(7.2)   96.2(6.6)  94.5(8.4)   91.3(8.8)
No. of pups born(%) 13.7(3.1)  15.0(1.7)  15.0(1.8)  15.1(2.7)
Delivery index(%) b) 87.6(15.4)  90.3(6.8)   94.1(7.2)  92.2(9.6)
Live pups born No. 13.3(2.9) 14.7(2.0)  14.9(2.0)  15.0(2.7)
Live birth index(%) c) 97.1(5.6) 97.8(3.6)   99.2(2.6)  99.4(2.1)
Sex ratio(M/F) 1.09(0.69) 1.05(0.50)  1.17(0.75)   0.76(0.44)
Dead pups born No. 0.5(0.9)  0.3(0.5)  0.1(0.3) 0.1(0.3)
Gestation length(day) 22.7(0.5)  22.7(0.5) 22.5(0.5)  11.6(0.5)
Gestation index(%) d) 100 100 100 100
Nursing index(%) e) 100 100 100 100
Live pups on day 4 No. 13.2(2.8)  14.6(2.1)  14.4(2.9) 14.5(2.9)
Viability index(%) f) 99.5(1.8)  99.3(2.3)  95.6(11.5)  96.7(6.7
Body weight of pups(g)        
Male Day 0 7.32(0.77)  7.13(0.52)  6.69(0.55)  6.87(0.84)
Male Day 4 11.71(1.76) 11.09(0.93)  10.23(0.98)*  10.60(1.47)
Male Day 0-4, gain(g) 4.39(1.04)  3.96(0.53) 3.54(0.77)*  3.73(0.80)
Male Body weight gain(%) g) 59.41(8.87)   55.54(6.16)  53.19(11.91)  54.39(9.50)
Female Day 0 6.93(0.83)  6.63(0.64) 6.33(0.58) 6.58(0.62)
Female Day 4 11.08(1.71)  10.28(1.01) 9.84(1.01)*  10.03(1.46)
Female Day 0-4, gain(g) 4.16(1.00)  3.65(0.56)   3.14(0.79)*  3.46(0.96)
Female Body weight gain(%) 59.63(10.42)  55.24(8.07)  49.95(13.09)  52.17(11.10)
         
Values are expressed as Mean (S.D.)
Significantly difference from 0 mg/kg group ; p < 0.05
a): (No. of implantation sites/no. of corpora lutea) x 100
b): (No. of pups born/no. of implantation sites) x 100
c): (No. of live pups born/no. of pups born) x 100
d): (No. of females with live pups delivered/ no. of pregnant remales) x 100
e): (No. of females nursing live pups/no. of females with normal delivery) x 100
f): ( No. of live pups on day 4/ no. of live pups born) x 100
g): ( Body weight gain/body weight on day 0) x 100
 

Applicant's summary and conclusion

Conclusions:
The reproductive NOAEL for the test material was determined to be >1000 mg/kg bw/day, the highest dose tested, for male and female rats (P generation). The developmental NOAEL was determined to be >1000 mg/kg bw/day, the highest dose tested, for the F1 generation as no fetotoxic, developmental, or teratogenic effects were observed.

Classification as a reproductive or developmental toxicant is not warranted under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP) or under Directive 67/548/EEC for dangerous substances and Directive 1999/45/EC for preparations.
Executive summary:

Male Crj: CD(SD) rats were treated with the test material for 46 days starting from 2 weeks prior to mating; female Crj: CD(SD) rats were treated with the test material starting from 2 weeks prior to mating to day 3 of lactation. Male amd female rats were cohabitated for up to 14 days or until proof of pregnancy was observed. Clinical observations, body weight, and food/water consumption were observed during the experimental period. Several indices for reproductive and for developmental performance were evaluated. Both male and female rats as well as the resulting pups were sacrificed and examined for gross pathology and for histopathological changes to the reproductive organs.

 

The reproductive NOAEL for the test material was determined to be >1000 mg/kg bw/day, the highest dose tested, for male and female rats (P generation). The developmental NOAEL was determined to be >1000 mg/kg bw/day, the highest dose tested, for the F1 generation as no fetotoxic, developmental, or teratogenic effects were observed.

 

Classification as a reproductive or developmental toxicant is not warranted under the new Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP) or under Directive 67/548/EEC for dangerous substances and Directive 1999/45/EC for preparations.