Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
one-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March to August 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well documented study performed according to OECD guideline
Qualifier:
according to
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
Version / remarks:
adopted May 26, 1983
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
The purity of the material used for the test was lower than the purity of the actual material. It contained ca. 26% of isomers of dimethyl tribenzenes. However, based on comparable basic structure of the impurities and the assumption that any toxic effects would increase with higher molecular weight and lipophilicity, we regard that the studies conducted with the lower purity material being "worst case" and can be used for the evaluation of the actual product.
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 6 wks (males) and 8 wks (females)
- Weight at study initiation: Males: 208-261 g; Females: 188-243 g
- Fasting period before study: no
- Housing: single in Macrolon cages (except during mating and lactation)
- Diet: "Ssniff R" pelleted diet produced by Ssniff Spezialitätendiäten GmbH, Soest, Germany; ad libitum
- Water: tap water; ad libitum
- Acclimation period: 12 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 +/- 1.5
- Humidity (%): 50-70
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: 0.5 % Sodium Carboxymethylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
All dosing solutions were prepared freshly each day by means of an ultraturrax. The emulsions were permanently stirred in the animal room prior to administration in order to maintain homogeneity.


VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: according to the dose levels 0, 2, 12 and 72 g/l, respectively
- Amount of vehicle (if gavage): 10 ml/kg bw
- Lot/batch no. (if required): 109 F 0361 (supplied by Sigma Deisenhofen)
- Purity: 0.5 % aqueous solution
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: 15 hours
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- After 21 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): singly in Macrolon cages
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dichloromethane extracts of the dose suspensions were analysed by photometry.
Additionally the stability of test substance in dose suspensions were analysed over an 24 hour interval.
Duration of treatment / exposure:
Males were treated for 10 weeks prior to mating and throughout mating up to sacrifice.
All females were treated for 2 weeks prior to mating and throughout mating, gestation and lactation up to weaning on, or shortly after day 28 post partum. Treatment ceased on the day prior to sacrifice.
Frequency of treatment:
once daily
Remarks:
Doses / Concentrations:
20 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
120 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
720 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
A preliminary experiment was carried out in pregnant rats with dose levels of 100, 500 and 1000 mg/kg bw. During the course of this study a clear effect upon weight development was noted during the first week of treatment of pregnant rats at all dose levels. Food consumption and food conversion ratios were also effected.
During a 3-month oral toxicity study in rats increased liver and kidney weights were found at dose levels of 100 and 500 mg/kg. The high dose of 720 mg/kg was selected to obtain clear toxic effects according to the recommendation of the OECD guideline. The low and mid doses were chosen in order to obtain information about possible dose-related effects and in order to establish a clear "no observable adverse effect level".
Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: No data


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily observations. Viability was checked twice daily. Dose response examinations were carried out at appropriate intervals after administration of the test article.


BODY WEIGHT: Yes
- Time schedule for examinations:
Males: Males were weighed prior to commencement of treatment, at weekly intervals during pre-mating treatment and after the mating period and at terminal sacrifice.

Females: All females were weighed prior to commencement of treatment and at weekly intervals during the pre-mating treatment phase. Weights were also recorded on days 0, 7, 14 and 20 of gestation; dams that littered were weighed on day 0 or 1 and on days 4, 7, 14, 21 and 28 post partum.
For non-mated females, weekly body weight determinations were continued until sacrifice, beginning on the first day after the mating period.

During mating, both males and females were weighed at least weekly for dose adjustment purposes; these body weights were not recorded. During gestation and lactation females were weighed daily for dose adjustment.


FOOD CONSUMPTION: Yes
- Time schedule for examinations:
Males: Food intake was determined at weekly intervals during pre-mating treatment until terminal sacrifice.

Females: Food intake was determined at weekly intervals during the pre-mating treatment phase. Food intake was also recorded on days 7, 14 and 20 of gestation; dams that littered were weighed on day 0 or 1 and on days 4, 7, 14, 21 and 28 post partum.
For non-mated females, weekly body weight determinations were continued until sacrifice, beginning on the first day after the mating period.

Food consumption was not determined during mating.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, age of developmental stage (pinna folding, hair growth, eye opening, upper incisor eruption), testis descent, surface righting reflex, startle reflex, air righting reflex, pupil reflex, ophthalmoscopy, special clinical examinations (modified Irwin Screening); between days 25 and 28 post partum behavioural examinations were carried out in at least 10 male and 10 female pups of each group (rotating rod test, inclined plane test, open field test)


GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals at the end of the mating period
- Parent females: All surviving animals on, or shortly after, day 28 of lactation, or after death of the whole litter
Females that failed to mate successfully or which were considered to have mated successfully but failed to produce a viable litter were sacrified approx. 26 days after the last day of the mating period.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the internal viscera.
Females that failed to mate successfully were examined macroscopically for organ changes with special attention to the organs of the reproduction tract.

HISTOPATHOLOGY/ORGAN WEIGHTS
Males: liver, testes with epididymides, seminal vesicles with coagulating gland, prostate, combined weights of kidneys (only in control and high dose group animals). These organs, together with the pituitary gland and any tissues showing severe changes, were preserved.
Females: Weights of liver and combined kidneys were determined only in control and high dose group. Ovaries, uterus, cervix, vagina, pituitary gland and tissues showing severe changes, were preserved.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 28 days of age.
- These animals were subjected to postmortem examinations as follows:


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations


HISTOPATHOLOGY / ORGAN WEIGTHS
Where possible, two male and two female pups were selected from each litter in order to determine the weight of brain, heart, liver, kidneys and spleen except the weight of liver in females of low and mid dose group (due to operational reasons).
Tissue showing severe changes were preserved.
Statistics:
Body weight changes, food consumption, pup weights and the number of implantations and offspring were subjected to analyses of variance with a subsequent multiple range test, or, if indicated, group mean values were compared by the "Kruskal-Wallis test" and "Mann-Whitney-U-test".
The quotient (weight changes/food consumtion) x 100 was calculated for each determination phase (food conversion ratio)
Organ weights were evaluated as both absolute and relative weights (% body weight). Dose groups were compared to the control group by the Dunnett test (two-tailed), modified according to Kramer.
Sex distribution, gestation length, the number of days until successful mating, physical and reflex development and behaviour in the rotating rod, inclined plane and open field tests were analysed by "Kruskal-Wallis test" and "Mann-Whitney-U-test".
Indices were compared by an appropriate statistical method, if indicated.
Where appropriate, the basic unit for all parameters was the litter.

References:
- Winer, B. J. (1971) Statistical principles in experimental design, International student edition, McGraw Hill Kogakusha Ltd.
- Snedecor, G. W. and W. G. Cochran (1967) Statistical methods. 6th ed. Iowa State University Press Ames, Iowa.
- Dunnett, C. W. (1955) A multiple comparison procedure for comparing several treatments with a control. J. Am. Stat. Assoc. 50, 1096-1121.
- Kramer, C. Y. (1956) Extension of multiple range test to group means with unequal numbers of replications. Biometrics 12, 307-310.
Reproductive indices:
Mating Index = 100 x No. of females in which mating was confirmed/No. of paired females
Fertility Index= 100 x No. of pregnant females/No. of paired females
Conception Rate= 100 x pregnant females/No. of females in which mating was confirmed
Abortion rate = 100 x No. of dams showing abortion/No. of pregnant dams
Gestation Index = 100 x No. of litters with live fetuses/No. of pregnant females
Pre-birth Loss Index = 100 x (No. of implantations - total litter size at birth)/No. of implantations
Pup Loss Index (at birth)= 100 x (total litter size at birth - live litter size at birth)/Total litter size at birth
Cumulative Pup Loss Index (Day x)= 100 x (total litter size at birth - live litter size on day x)/Total litter size at birth
Sex Ratio= 100 x No. of live male pups per litter/No. of live pups per litter
Clinical signs:
effects observed, treatment-related
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:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No treatment-related clinical findings were noted in males and females of all dose groups except salivation which was observed in males in the high dose group during pre-mating only in two animals and nearly all animals after the mating period. Salivation was observed in females during gestation from day 8 onwards in high dose group and during lactation in mid and high dose group with dose-related increase in incidence. Salivation is considered to be a local effect of the test article.
Two males of the control group showed dermatopathia on the neck from weeks 2 and 3 onwards which was treated with dexpanthenol ointment from week 5 until week 7 or 8, respectivlely. The the animals displayed alopecia only.
One female of the high dose group appeared emaciated during the last weeks of lactation.
One male of the mid dose group was found dead at the end of week 13 of treatment. This spontaneous death is considered to be without toxicological importance. One female of high dose group was found dead on day 24 of lactation.

BODY WEIGHT (PARENTAL ANIMALS)
Weight gain rates of high dose males were significantly reduced during the entire treatment period (see table 1). In females during the pre-mating period reduced body weight gain was observed in mid and high dose group but attained significance only in the high dose group. During gestation decrease in weight gain of high dose females was observed between days 0 and 7 and 14 and 20 and over the whole period of gestation. Between beginning and end of lactation weight gain of high dose females was significantly increased relative to control females. They showed a smaller weight loss from days 14 to 21 and 21 to 28 compared to controls resulting in higher absolute body weights of high dose females compared to control. The increased weight gain was probably a compensation of decreased body weight at delivery as the mean body weigth of the high dose group females were decreased at the start of lactation and only slightly increased on day 28 compared to control females. (see table 2 and 3)

FOOD CONSUMPTION AND FOOD CONVERSION RATIO (PARENTAL ANIMALS)
No significant changes in food consumption was found in males and females during pre-mating treatment. During gestation food consumption was significantly reduced in high dose females between days 1 and 7 and over the entire period of gestation. During the lactation period mean food consumption values were slighly decreased in high dose females on days 14, 21 and 28 of lactation but with a high standard deviation. In addition the pups also eat the provided food particularly in the last part of lactation and the number of pups per dam was decreased in high dose group.
Food conversion ratio of males showed only slighly decreased mean values in high dose group in weeks 3, 4, 6, 7, and 8 of treatment. This corresponds to the reduced weight gain in this dose group. In females the food conversion ratio was decreased in high dose females during week 1 of pre-mating treatment period and slightly on day 20 of gestation.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Mating index, mating performance, fertility index and conception rate did not reveal any differences attributable to the treatment. The fertility index and conception rate of the high dose group were decreased but both were within the historical range of historical data and the differences were not statistically significant.
No females showed signs of abortion or premature delivery. Gestation indices did not reveal treatment-related differences. The decreased value of the high dose group was due to one animal only. No significant differences were found for gestation lenghts. No instances of prolonged parturition or dystocia. No significant differences were found either in the total number of implantations or in the distribution between the right and left uterine horns. Pre-birth loss indices did not differ significantly between groups and no-doserelationship was evident.

ORGAN WEIGHTS (PARENTAL ANIMALS)
The relative and absolute liver weights were significantly increased in males of all dose groups with a clear dose-relationship and in females only in the high dose group. Absolute and relative kidney weights were significantly increased in males of the high dose group. In females absolute kidney weights were significantly increased in the high dose group. However since the body weights of these females were higher the relative weights did not reveal a significant difference. Thus, an effect on the weights of kidneys cannot be determined for the females. (see table 4)
With respect to the reproductive organs, relative prostate weights were significantly increased in high dose males. Since mating performance and fertility did not reaveal any treatment-related differences, this finding may be of minor toxicological importance. The weights of testes and seminal vesicles did not show any significant differences. (see table 4)

GROSS PATHOLOGY (PARENTAL ANIMALS)
No distinct treatment-related findings were found during necropsy of both males and females.
Key result
Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day (actual dose received)
Sex:
male
Basis for effect level:
other: based on body weight; food consumption; organ weights; liver weight effects appear to be an adaptive response rather than a toxic effect;
Key result
Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day (actual dose received)
Sex:
female
Basis for effect level:
other: body weight; food consumption; organ weights
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
ca. 720 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: absence of effects on reproductive functions
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
LITTER SIZE
The litter size was significantly reduced in the high dose group on days 4, 7, 14, 21 and 28 post partum which was predominantly caused by the increased pup loss after birth. (see table 5)

SEX DISTRIBUTION, SEX RATIO (OFFSPRING)
The sex ratio did not reveal important differences. The percentage of males showed only slight intergroup variation, which was considered to be within the physiological range.

VIABILIY INDEX (OFFSPRING)
The pup loss index at birth was not affected by treatment in all test groups. Correspondingly, the number of litters with dead pups at birth was very similar between all groups.
Cumulative pup loss indices were increased in the high dose group from day 7 post partum onwards. (see table 6)
The cumulative pup loss index of the mid dose group also seemed to be increased. But an evaluation of the individual mid dose group litters affected showed that the increased cumulative pup loss index was predominantly caused by one litter, in which 15 of 16 pups were found dead. Additionally with respect to the number of litters with dead pups, an increase in the mid dose group cannot be determined.

CLINICAL SIGNS (OFFSPRING)
No clear indication of treatment effect on the appearance and general condition of offspring.

BODY WEIGHT (OFFSPRING)
Mean group body weights of both male and female pup were significantly reduced in the high dose group at birth and on days 4, 7, 14, 21 and 28 post partum. (see table 7)

ORGAN WEIGHTS (OFFSPRING)
Relative weights of spleen did not reveal any significant differences between the groups. Relative heart weights were significantly increased in high dose group males and females. Decreased relative heart weights in males of the the low dose group is considered to be not treatment-related, since relative heart weights in mid dose group are lower (no clear dose-relationship) and no increase of relative heart weights was found in females of the low dose group.
Relative kidney weights and relative brain weights were significantly increased in high dose males and females. Decreased relative brain weights in males and females of the low dose group is considered to be not treatment-related as the brain weights of the mid dose group were close to those of the control (no clear dose-relationship). Also the significant difference in relative liver weights of the low dose females is considered to be incidental since no dose-relationship existed. (see table 8)

GROSS PATHOLOGY (OFFSPRING)
Necropsy of pups found dead during pre-weaning development did not reveal any specific findings which point to a manifestation of parental treatment-effect.
Macroscopic examinations of pups sacrificed at weaning revealed no changes clearly attributable to the treatment of parents. A diaphragmatic hernia was found in one pup of the high dose group. This finding is believed to be associated with the distinct growth or developmental retardation found in this dose group.

PHYSICAL DEVELOPMENT (OFFSPRING)
No obviously treatment-related retardations were found for the live pups. Group mean values of cumulative frequencies of pups which had attained each specific developmenal stage were comparable between the groups with the exception of hair growth. A slightly delayed commencement of hair growth was observed in the high dose group, as the cumulative frequency on days 5 and 6 was decreased relative to control. Although not statistically significant, this decrease corresponds to the reduction of group mean body weights in the high dose pups.
The occasional significant differences in the low and mid dose group are considered to be incidental.

REFLEX DEVELOPMENT (OFFSPRING)
The onset of the startle reflex was definitely retarded in the high dose group pups; the cumulative frequency of responding pups was significantly decreased relative to control. All pups of the high dose group showed this reflex only on day 18 post partum whereas control pups did on day 15 post partum. This retardation was most probably in corrspondence to the reduced body weight development.
Evaluation of the development of the other reflexes examined did not reveal any treatment-related differences.

OTHER FINDINGS (OFFSPRING)
During ophthalmoscopic examination of pups after day 18 post partum only a few spontaneous findings were ascertained.
The modified Irwin Screening of live pups did not provide evidence of a treatment-related effect.
The inclined plane test showed a significantly decreased clinging ability of high dose group female pups. A slightly but non-significantly decreased clinging ability may also be stated for high dose group male pups. This decrease seems to be in correspondence with the affected weights of the brain and the developmental retardations which were found in high dose group pups.
Results of the open field test did not provide clear evidence of a treatment-related effect. Since no clear dose-relationship is obvious a treatment-related effect cannot be stated.
No significant differences were found during the rotating rod test in both male and female pups.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
720 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: all effects observed at the high dose level (e.g. litter size; pup weight;) are linked to the body weight reduction of the pups itself and appear to be secondary to maternal toxicity.
Reproductive effects observed:
not specified
Table 1: Weight changes (parental males; mean values and standard deviations)
Group Control 20 mg/kg 120 mg/kg 720 mg/kg
Weeks 0-15  absolute (g) 383.3 ± 48.3 385.2 ± 50.3 390.5 ± 38.9 346.0 ± 47.0*
relative (%) 100.0 100.5 101.9 90.3
Weeks 5-10 absolute (g) 105.8 ± 20.1 101.2 ± 18.5 101.6 ± 20.5 87.8 ± 23.0*
relative (%) 100.0 95.7 96.1 83.1
Weeks 10-15  absolute (g) 48.4 ± 17.8 49.7 ± 13.9 45.2 ± 20.3 34.7 ± 14.0*
relative (%) 100.0 102.7 93.3 71.6
Weeks 13-15  absolute (g) 26.3 ± 11.0 20.3 ± 10.9 25.1 ± 15.7 19.3 ± 8.4*
relative (%) 100.0 77.5 95.6 73.5
*p < 0.05 (Scheffé test)
Table 2: Weight changes (parental females; mean values and standard deviations)
Group Control 20 mg/kg 120 mg/kg 720 mg/kg
Weeks 0-2 (pre-mating)  absolute (g) 39.1 ± 10.5 37.9 ± 12.2 34.6 ± 7.9 31.4 ± 7.1*
relative (%) 100.0 96.8 88.5 80.3
Days 0-20 (gestation)  absolute (g) 156.7 ± 22.4 150.1 ± 23.4 153.8 ± 23.7 133.7± 19.4*
relative (%) 100.0 95.8 98.2 85.3
Days 0-7 (gestation)  absolute (g) 37.6 ± 7.9 37.9 ± 7.1 38.6± 9.5 34.6 ± 7.0*
relative (%) 100.0 100.8 102.6 91.6
Days 14-20 (gestation)  absolute (g) 82.4 ± 12.2 80.9 ± 19.5 82.3 ± 20.2 70.5 ± 12.8*
relative (%) 100.0 98.2 99.9 85.6
Days 0/1-28 (lactation)  absolute (g) -25.8 ± 19.0 -18.7 ± 35.0 -22.0 ± 19.6 5.8 ± 16.8*
relative (%) 100.0 72.3 85.1 -22.3
Days 14-21 (lactation)  absolute (g) -26.6 ± 20.2 -24.0 ± 17.0 -15.2 ± 17.3 -4.6 ± 13.2
relative (%) 100.0 90.5 57.1 17.1
Days 21-28 (lactation)  absolute (g) -36.7 ± 19.3 -39.7 ± 21.8 -37.5 ± 17.2 -18.5 ± 14.4*
relative (%) 100.0 108.0 102.0 50.3
*p < 0.05 (Dunnett-test)
Table 3: Body weights of parental females during lactation (Mean values and standard deviations)
Group Control 20 mg/kg 120 mg/kg 720 mg/kg
Day 0/1 (lactation) 323.4 ± 25.6 313.7 ± 37.7 329.9 ± 28.6 304.2 ± 18.0
Day 4 (lactation) 338.0 ± 31.0 332.9 ± 22.4 344.0 ± 33.4 311.2 ± 18.1
Day 7 (lactation) 357.4 ± 30.3 351.2 ± 22.7 356.4 ± 28.5 327.2 ± 21.2
Day 14 (lactation) 360.9 ± 29.0 358.7 ± 23.1 360.6 ± 25.0 333.8 ± 23.6
Day 28 (lactation) 297.6 ± 25.6 295.0 ± 16.6 308.0 ± 28.2 311.4 ± 15.6
Table 4: Selected organ weights of parental animals (mean values and standard deviations)
Group Control 20 mg/kg 120 mg/kg 720 mg/kg
Liver weights males 23.2310 ± 3.0147 25.7816 ± 4.1249* 27.8573 ± 2.7969* 29.5665 ± 4.4976*
absolute (g) females 16.0065 ± 1.9983 22.7884 ± 2.7078*
Liver weights males 3.771 ± 0.361 4.137 ± 0.430* 4.456 ± 0.302* 5.089 ± 0.436*
relative (%) females 5.340 ± 0.524 7.395 ± 0.395*
Kidney weights males 4.1578 ± 0.5618 4.8922 ± 0.6641*
absolute (g) females 2.7601 ± 0.2707 3.0071 ± 0.3162*
Kidney weights males 0.675 ± 0.071 0.845 ± 0.087
relative (%) females 0.922 ± 0.079 0.977 ± 0.100
Prostate weights absolute (g) 1.6846 ± 0.1821 1.6622± 0.2078 1.7406 ± 0.2621 1.7579 ± 0.2005
relative (%) 0.2752 ± 0.0347 0.2689 ± 0.0340 0.2801 ± 0.0488 0.3057 ± 0.0419*
*p < 0.05 (Dunnett-test, two-tailed)
Table 5: Litter size; No. of pups (mean values and standard deviations)
Group Control 20 mg/kg 120 mg/kg 720 mg/kg
at birth (total) 15.0 ± 2.8 15.3 ± 2.9 14.4 ± 3.4 13.6 ± 3.6
at birth (live pups) 14.9 ± 2.8 15.2 ± 3.0 14.1 ± 3.3 13.4 ± 3.8
Day 4 14.7 ± 2.8 15.1 ± 3.0 13.4 ± 4.1 12.8 ± 3.8*
Day 7 14.5 ± 2.7 14.9 ± 3.0 13.2 ± 4.1 12.2 ± 4.1*
Day 14 14.5 ± 2.7 14.9 ± 3.0 13.1 ± 4.1 12.1 ± 4.3*
Day 21 14.5 ± 2.7 14.9 ± 3.0 13.1 ± 4.1 11.9 ± 4.3*
Day 28 14.5 ± 2.7 14.9 ± 3.0 13.1 ± 4.1 11.9 ± 4.4*
number of litters 21 23 22
* p (U-test) < 0.05
Table 6: Cumulative pup loss index (%) 
Group Control 20 mg/kg 120 mg/kg 720 mg/kg
Day 4 2.8 1.1 7.0 5.8
Day 7 3.8 2.3 8.2 10.4
Day 14 4.1 2.6 8.5 11.6
Day 21 4.1 2.6 8.5 12.4
Day 28 4.1 2.6 8.5 12.7
Tabe 7: Body weight of offspring (g; mean values and standard deviations)
Group Control 20 mg/kg 120 mg/kg 720 mg/kg
males
Day 0/1 6.75 ± 0.48 6.44 ± 5.6 6.78 ± 0.72 6.19 ± 0.53*
Day 4 10.02 ± 1.08 9.64 ± 1.22 9.91 ± 1.79 8.34 ± 1.36*
Day 7 14.52 ± 1.81 13.95 ± 1.95 14.59 ± 2 08 11.76 ± 2.14*
Day 14 26.69 ± 4.22 25.56 ± 3.82 26.40 ± 3.86 21.90 ± 3.77*
Day 21 45.60 ± 4.22 42.86 ± 7.49 44.65 ± 7.14 35.59 ± 7.26*
Day 28 78.32 ± 9.91 73.79 ± 12.62 78.88 ± 11.37 65.86 ± 12.71*
females
Day 0/1 6.33 ± 0.42 6.18 ± 0.56 6.43 ± 0.63 5.94 ± 0.51*
Day 4 9.46 ± 0.91 9.17 ± 1.24 9.45 ± 1.40 8.03 ± 1.33*
Day 7 13.70 ± 1.58 13.17 ± 2.05 13.75 ± 2.36 11.38 ± 2.03*
Day 14 25.55 ± 4.26 24.25 ± 4.17 24.98 ± 4.26 21.07 ± 3.71*
Day 21 43.38 ± 6.07 41.8 ± 7.70 42.51 ± 6.96 34.07 ± 7.26*
Day 28 73.57 ± 9.16 68.99 ± 12.60 73.17 ± 11.38 61.85 ± 12.18*
* p< 0.05 (Dunnett-test)
Table 8 Selected relative organ weights of offspring (%, mean values and standard deviations)
Group Control 20 mg/kg 120 mg/kg 720 mg/kg
Heart  males 0.5396 ± 0.0754 0.5858 ± 0.0810* 0.5727 ± 0.0808 0.6135 ± 0.0750*
females 0.5357 ± 0.0688 0.5701 ± 0.0793 0.5770 ± 0.0930 0 5823 ± 0.0604*
Kidney males 1.419 ± 0.120 1.462 ± 0.158 1.421 ± 0.120 1.551 ± 0.141*
females 1.443 ± 0.102 1.468 ± 0.315 1.463 ± 0.103 1.536 ± 0.147*
Brain  males 1.9647 ± 0.2751 2.141 ± 0.3488* 2.0290 ± 0.3020 2.3799 ± 0.4206*
females 2.0406 ± 0.2921 2.1350 ± 0.3288 2.1193 ± 0.3733 2.3916 ± 0.4123*
Liver  males 5.967 ± 0.512 5.762 ± 0.494 5.621 ± 0.877 6.127 ± 0.398
females 6.092 ± 0.562 5.793 ± 0.550* 5.942 ± 0.470 6.149 ± 0.451
*p < 0.05 (Dunnett-test, two-tailed)
Conclusions:
The NOAEL for general, systemic toxicity for males is 120 mg/kg/d, based on increased kidney and prostate weights and reduced body weights. The increase in liver weights is likely to be an adaptive response to the increased metabolic activity of the liver and can not be regarded as a toxic effect.

The NOAEL for general, systemic toxicity for parental females is also 120 mg/kg bw /d based on reduced body weight gain, reduced food consumption and increased organ weights (liver, and kidney) in the high dose group.

The NOAEL for reproductive toxicity is considered to be 720 mg/kg bw /d for the parental rats since no changes in reproductive parameters was observed.

The NOAEL for developmental toxicity in the F1 progeny is 720 mg/kg bw/d since effects on litter size, pup weight, pup survival and organ weights are secondary to maternal toxicity and reduced pup weight.
Executive summary:

The effects on fertility of the test material was evaluated in an OECD 415 one-generation study in rats. Animals received the test material via oral gavage in doses up to 720 mg/kg/d. Treatment related effects included organ weight effects (kidney & liver; males all dose groups and females high dose group only) and reduced food intake. Prostate weights were increased in high dose males as well.

No effects on reproductive parameters were observed. Mating performance and fertility were unaffected up to a dose level of 720 mg/kg. In the high dose group litter size, pup weight, pup survival and organ weights were affected, but these effects are secondary to maternal toxicity.

Effect on fertility: via oral route
Dose descriptor:
NOAEL
720 mg/kg bw/day
Additional information

Dibenzylbenzene, ar-methyl derivative was evaluated for effects on fertility in a one-generation study in rats. No treatment related specific effects on reproduction was observed. Findings observed in the study were either slight and/or can be related to the maternal toxicity caused by treatment. No effects on reproductive parameters were observed. Mating performance and fertility were unaffected up to a dose level of 720 mg/kg. In the high dose group litter size, pup weight, pup survival and organ weights were affected.

NOAEL reproductive toxicity: 720 mg/kg/d, since no effects on reproductive parameters were observed.

NOAEL embryotoxicity: 720 mg/kg/d, since all effects observed in the highest dose group appear to be linked to the body weight reduction of the pups itself linked to maternal toxicity.

NOAEL parental toxicity: 120 mg/kg/d, based on bodyweight effects, food intake and kidney and prostate weights; liver weight effects can be regarded as adaptive response linked to metabolic activation.


Short description of key information:
Dibenzyltoluene was evaluated for effects on fertility in a one-generation study in rats. No treatment related specific effects on reproduction was observed. Findings observed in the study were either slight and/or can be related to the maternal toxicity caused by treatment.

Effects on developmental toxicity

Description of key information

The effects of Dibenzylbenzene, ar-methyl derivative on the development was evaluated in two separate developmental toxicity study in rats.

No treatment related specific effects on the development of fetuses were observed.

Findings observed in the studies were either slight and/or can be related to the maternal toxicity caused by treatment.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 1992 June 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: well documented study performed according to OECD guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
once-only false dosing of 17 animals in low dose group
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
The purity of the material used for the test was lower than the purity of the actual material. It contained ca. 26% of isomers of dimethyl tribenzenes. However, based on comparable basic structure of the impurities and the assumption that any toxic effects would increase with higher molecular weight and lipophilicity, we regard that the studies conducted with the lower purity material being "worst case" and can be used for the evaluation of the actual product.
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: about 8 weeks
- Weight at study initiation: 197 to 280 g
- Fasting period before study: no
- Housing: single, in Macrolon cages
- Diet : "Ssniff R" pelleted diet by Sniff Spezialdiäten GmbH, Soest, Germany; ad libitum
- Water: tap water ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 °C +/- 1.5 °C
- Humidity (%): 50 - 70 %
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: April 23, 1992 To: June 2, 1992
Route of administration:
oral: gavage
Vehicle:
other: 0.5 % Sodium Carboxymethylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Preparation of doses by homogenizing the test substance with the vehicle by means of ultraturrax, stirring constantly prior to administration in order to maintain homogeneity.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: according to the dose levels 2.5, 15 and 100 g/l, respectively
- Amount of vehicle (if gavage): 10 ml/kg bw
- Lot/batch no. (if required): 109 F 0361 (supplier: SIGMA, Deisenhofen, Germany)
- Purity: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical verification of doses and stability control by photometry (at fixed wavelenght of 262 nm) after extraction of test substance from dose suspensions by dichloromethane.

Analyses of samples of dosing emulsions from all dose groups at commencement and termination of treatment. Samples of 100 ml per group were drawn and analyses for identity and concentration were performed. The allowance limit of concentration was +/- 10%.
The stability and homogeneity of the test article in the vehicle had been proven by analysing a representative sample at different intervals after preparation during dose range finding study.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/2
- Length of cohabitation: overnight
- Further matings after two unsuccessful attempts: no data
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
from day 6 to day 15 of gestation
Frequency of treatment:
once daily
Duration of test:
until day 20 of gestation
Remarks:
Doses / Concentrations:
25 mg/kg
Basis:
actual ingested
; 17 animals were treated erroneously once with dose of 150 mg/kg either on day 6 or 7 of gestation
Remarks:
Doses / Concentrations:
150 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Preliminary dose range finding was conducted with dose levels of 100, 500 and 1000 mg/kg. The test substance was administered to 8 female rats per dose once daily by gavage from thoughout gestation and lactation up to day 4 post partum. Observations: Significantly decreased weight gains during the first week of treatment (days 0 to 7 of gestation) in pregnant rats at all dose levels. Food consumption was significantly decreased in the high dose group from days 1 to 7 and 1 to 20 of gestation. Food conversion ratios were decreased in all dose groups on day 7 of gestation. The pre-birth loss index in the high dose group was increased. In the F1 generation in the high dose group the mean litter size was significantly reduced at birth and at day 4 post partum and in correspondence, the number of males was significantly decreased. The pup loss index at birth and the number of litters with dead pups was found to be increased in the high dose group. Rationale: The dose level of 1000 mg/kg was selected according to the recommendations of the OECD guideline as high dose level resulting in toxic effects. The low and mid dose group were chosen to obtain information about possible dose-related effects and in order to establish a clear NOAEL.
- Animal assignment: random
Maternal examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily over the entire period of the investigation, viability twice daily
Special regard on sensory and motor behaviour, coat, urine and fecal excretion, condition of body orifices and signs of ill health. Additionally, during the treatment period, dose rsponse examinations were carried out at appropriate intervals after administration of the test article. During gestation females were observed closely for signs of abortion or premature delivery.

BODY WEIGHT: Yes
- Time schedule for examinations: daily but recorded only on days 0, 6, 10, 15 and 20 of gestation
Body weight development was evaluated over the following phases: 0-6, 6-15, 15-20 and 0-20 days

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes (on days 0, 6, 10, 15 and 20 of gestation)
- Food consumption for each animal determined: Yes
- Mean daily diet consumption was calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 20 of gestation
- Organs examined: macroscopic examination of internal viscera (not specified)

OTHER:
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: location of implantation sites; number and location of live and dead fetuses, weight of fetuses and placentae, head/tail presentation of fetuses, uteri ore individual uterine horns without visible implantations were stained to reveal evidence of embryonic death at early stages of implantation
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter (in-situ dissection modified according to H. Sterz (1977) ["Routine examination of rat and rabbit fetuses for malformation of internal organs: Combination of Barrow's and Wilson's methods, in: D. Neubert, H.-J. Merker and T. E: Kwasigroch (eds) Methods in prenatal toxicology, Thieme Publishers, Stuttgart, pp. 113-122])
- Skeletal examinations: Yes: half per litter (skeletal structures were stained with alizarin red S according to Kawamura et al. [Kawamura, S. , A. Hirohashi, T. Kato and M. Yasuda (1990) Bone staining technique for fetal rat specimens without skinning and removing adipose tissue, Cong. Anom. 30, 93-95.
- Head examinations: No data
- Other: sex determination by ano-genital distance
Statistics:
Analyses of variance with a subsequent multiple range test for body weight changes, food consumption and reproductioons parameters, or, if indicated, group mean values were compared by the "Kruskal-Wallis test" and "Mann-Whitney-U-test". The maternal body weight on day 20 of gestation was corrected for gravid uterine weight and the corrected rate of body weight gain (days 0-20 of gestation) was calculated.
Indices were compared statistically by the "Mann-Whitney-U-test".
References:
- Winer, B. J. (1971) Statistical principles in experimental design, International student edition, McGraw Hill Kogakusha Ltd.
- Snedecor, G. W. and W. G. Cochran (1967) Statistical methods. 6th ed. Iowa State University Press Ames, Iowa.
Indices:
Abortion rate = 100 x No. of females with abortion/Total No. of pregnant females
Resorption rate = 100 x No. of resorptions/No. of imlantations
Pre-implantation Loss Index = 100 x No. of corpora lutea - No. of implantations/ No. of corpora lutea
Post-implantation Loss index = 100 x No. of implentations - No. of implantations/ No. of implantations
Live Birth Index = 100 x No. of live fetuses/total No. of fetuses
Runts Index = 100 x No. of runts/Total No. of runts
Variation Index = 100 x No. of live fetuses with variations/total No. of live fetuses examined
Anomaly Index = 100 x No. of live fetuses with variations/total No. of live fetuses examined
Malformation Index = 100 x No. of live malformed fetuses/total No. of live fetuses examined
Historical control data:
Recent historical data containing findings of visceral and skeletal examinations of fetuses derived from females of the control groups.

Number of fetuses/litters examined:
Visceral abnormalities:
Head: 1506 fetuses/218 litters; other soft tissue: 145 fetuses/22 litters
Skeletal examination: 1593 fetuses
Skeletal abnormalities: 1593 fetuses/218 litters
Variations and anomalies: 1446 fetuses/197 litters
Clinical signs:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Body weight gain:
Dose related significantly decreased body weight gain from gestation days 6 to 15 at 150 and 1000 mg/kg bw . At 1000 mg/kg bw also significantly decreased body weight gain throughout the entire gestation period (days 0 to 20). At 150 mg/kg bw decreased body weight gain throughout gestation days 0 to 20 but not statistically significant (see Tables 1 and 2). Corrected body weights (body weight on gestation day 20 minus gravid uterine weigth) were slightly decreased at 150 and 1000 mg/kg bw but did not reveal significance (see Table 3).
The weight gain of the animals of the low dose group which were treated erroneously once with the mid dose on day 6 or 7 of gestation was increased from day 6 to 10 and day 6 to 15 of gestation compared to the 8 "right-treated" animals of the low dose group (see Table 4). There is no indication that the wrong dosing had any impact on the outcome of the study.
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Fetal weights:
Mean fetal body weights were significantly reduced in both males and females at 1000 mg/kg bw. Also the combined group of males and females at this dose showed a significant reduction (see Table 5).


Number of runts/runts index:
One runt each were found in the low and mid dose groups. 6 runts were found at 1000 mg/kg bw. Consequently the runts index was increased (see Table 6). The increased runts index is most likely to be in correspondence with the reduced fetal body weights found in high dose group.


Number of resorptions/resorption index:
Significantly increased total number of resorptions (both early and late resorptions) were observed at 1000 mg/kg bw (see Table 7). Consequently the resorption index was significantly increased in this group. The number of females without resorptions was decreased and the number of females with two or more resorptions was increased at 1000 mg/kg bw (see Table 8).

Post-implantation Loss index:
Since no other post-implantation losses apart from resorptions were found the post-implantation index is identical with the resorption index and significantly increased at 1000 mg/kg bw (Table 9).


Visceral examination:
Visceral examination of fetuses revealed diaphragmatic hernia in three fetuses at 1000 mg/kg bw, two of them in the same litter. A slightly increased incidence of dilated ureters was observed at 1000 mg/kg bw.


Skeletal examination:
Skeletal examination showed a slight increase of fetuses with less than 4 ossification centres and also an increase of the frequency of affected litters at 1000 mg/kg bw (Table 11). Though the incidence of affected fetuses was within the historical range the historical mean value was distinctly lower.
The incidence of cleaved sternebrae was slightly increased in the high dose group with respect to the affected litters (Table 12). This frequency was above the historical range. Also the number of fetuses with 3 ossified metacarpals was increased in the high dose group with a frequency of affected fetuses slightly above the historical range (Table 11).
Cleaved or dumpbell shaped thoracic vertebral bodies were clearly more frequently observed in fetuses at 1000 mg/kg bw. The frequency of both affected fetuses and litters were elevated. The incidence of cleaved vertebrae was distinctly above the historical range and that of the dumpbell shaped vertebrae was at the upper limit. A slightly dose-related trend of increased frequency is seen in at 150 mg/kg bw. (Table 12)
Non-ossification of coccygeal vertebrae was observed in a higher frequency in at 1000 mg/kg bw, the number of affected litters being slightly above the range of historical data. (Table 12)

These findings in the skeletal examination are considered to be signs of retarded fetal ossification and provide a further indication of developmental delay in fetuses of the high dose group.

The anomaly indices were increased with increasing doses. However, the anomaly index of the control group was low compared to those of historical studies. At 25 mg/kg bw both the anomaly index and the frequency of litters with anomalies were below the respective historical mean values. At 150 mg/kg the anomaly index was close to the mean historical value but the frequency of litters with anomalies was slightly increased. At 1000 mg/kg bw both parameters were distinctly above the respective historical mean values and at the upper limit of the historical range. (Table 13)

The visceral and skeletal malformation index in the high dose group was 1.6 % and was based on the occurrence of three different types of malformations, i. e. one skeletal and two different visceral ones. (Table 13)
Fetal weights:
Mean fetal body weights were significantly reduced in both males and females at 1000 mg/kg bw. Also the combined group of males and females at this dose showed a significant reduction (see Table 5).


Number of runts/runts index:
One runt each were found in the low and mid dose groups. 6 runts were found at 1000 mg/kg bw. Consequently the runts index was increased (see Table 6). The increased runts index is most likely to be in correspondence with the reduced fetal body weights found in high dose group.


Number of resorptions/resorption index:
Significantly increased total number of resorptions (both early and late resorptions) were observed at 1000 mg/kg bw (see Table 7). Consequently the resorption index was significantly increased in this group. The number of females without resorptions was decreased and the number of females with two or more resorptions was increased at 1000 mg/kg bw (see Table 8).

Post-implantation Loss index:
Since no other post-implantation losses apart from resorptions were found the post-implantation index is identical with the resorption index and significantly increased at 1000 mg/kg bw (Table 9).


Visceral examination:
Visceral examination of fetuses revealed diaphragmatic hernia in three fetuses at 1000 mg/kg bw, two of them in the same litter. A slightly increased incidence of dilated ureters was observed at 1000 mg/kg bw.


Skeletal examination:
Skeletal examination showed a slight increase of fetuses with less than 4 ossification centres and also an increase of the frequency of affected litters at 1000 mg/kg bw (Table 11). Though the incidence of affected fetuses was within the historical range the historical mean value was distinctly lower.
The incidence of cleaved sternebrae was slightly increased in the high dose group with respect to the affected litters (Table 12). This frequency was above the historical range. Also the number of fetuses with 3 ossified metacarpals was increased in the high dose group with a frequency of affected fetuses slightly above the historical range (Table 11).
Cleaved or dumpbell shaped thoracic vertebral bodies were clearly more frequently observed in fetuses at 1000 mg/kg bw. The frequency of both affected fetuses and litters were elevated. The incidence of cleaved vertebrae was distinctly above the historical range and that of the dumpbell shaped vertebrae was at the upper limit. A slightly dose-related trend of increased frequency is seen in at 150 mg/kg bw. (Table 12)
Non-ossification of coccygeal vertebrae was observed in a higher frequency in at 1000 mg/kg bw, the number of affected litters being slightly above the range of historical data. (Table 12)

These findings in the skeletal examination are considered to be signs of retarded fetal ossification and provide a further indication of developmental delay in fetuses of the high dose group.

The anomaly indices were increased with increasing doses. However, the anomaly index of the control group was low compared to those of historical studies. At 25 mg/kg bw both the anomaly index and the frequency of litters with anomalies were below the respective historical mean values. At 150 mg/kg the anomaly index was close to the mean historical value but the frequency of litters with anomalies was slightly increased. At 1000 mg/kg bw both parameters were distinctly above the respective historical mean values and at the upper limit of the historical range. (Table 13)

The visceral and skeletal malformation index in the high dose group was 1.6 % and was based on the occurrence of three different types of malformations, i. e. one skeletal and two different visceral ones. (Table 13)





Key result
Dose descriptor:
NOAEL
Effect level:
> 1 000 mg/kg bw/day
Basis for effect level:
reduction in number of live offspring
changes in sex ratio
fetal/pup body weight changes
Key result
Abnormalities:
not specified
Key result
Developmental effects observed:
not specified

Body weight gain:

Table 1. Body weight changes [g] (Analysis of variance, mean values, standard deviations)

 

days of gestation

group

0-6

6-15

15-20

0-20

0 mg/kg

37.1± 6.7

55.8 ± 5.8

80.0 ± 11.4

173.0 ± 15.7

25 mg/kg

40.8 ± 6.1

51.5 ± 8.3

79.8 ± 11.8

172.0 ± 18.5

150 mg/kg

35.3 ± 11.7

47.8x ± 8.3

78.2 ± 15.1

161.2 ± 21.9

1000 mg/kg

38.1 ± 8.9

40.5x ± 9.0

81.9 ± 15.0

160.5x ± 20.1

MSB

129.95

1 16.03

56.75

1095.58

MSW

74.36

63.03

180.16

368.17

F

1.75

16.12***

0.32

2.98*

DF

3/   93

3/   93

3/   93

3/   93

P

0.161

0.000

0.817

0.035

x  = p 0.05 (Dunnett-test); * = p 0.05 (F-test); *** = p 0.001 (F-test)

Table 2. Mean weight development [g] (mean values and standard deviations)

 

day of gestation 

group

0

6

10

15

20

0 mg/kg

225.1 ± 9.5

262.2 ± 11.2

285.0 ± 10.6

318.0 ± 12.4

398.0 ± 17.6

25 mg/kg

225.3 ± 13.8

266.1 ± 14.6

286.8 ± 15.0

317.6 ± 18.9

397.4 ± 26.3

150 mg/kg

231.3 ± 22.5

266.5 ± 21.1

284.3 ± 21.0

314.3 ± 23.8

392.5 ± 31.5

1000 mg/kg

234.0 ± 16.8

272.1 ± 18.4

286.6 ± 19.8

312.6 ± 19.5

394.5 ± 28.6

Table 3. Corrected body weights and weight changes [g] (Analysis of variance, mean values, standard deviations)

group

Day 20 (corrected)

Day 0-20 (corrected)

0 mg/kg

311.9 ± 13.1

86.8 ± 11.4

25 mg/kg

313.0 ± 18.8

86.7 ± 13.6

150 mg/kg

311.9 ± 22.5

80.6 ±15.5

1000 mg/kg

313.6 ± 22.3

79.6 ± 16.3

MSB

16.03

362.14

MSW

381.72

204.75

F

0.04

1.77

DF

3/93

3/93

P

0.989

0.159

Table 4. Weight changes [g] and mean weight development [g] of low dose group (25 mg/kg bw)  (Mean values and standard deviations)

 

Group 25 mg/kg

Body weights

a

b

Day 0

232.4 ± 19.0

222.0 ± 9.5

Day 6

272.1 ± 21.4

263.2 ± 9.5

Day 10

288.9 ± 25.8

285.8 ± 6.6

Day 15

322.3 ± 30.5

315.4 ± 10.5

Day 20

401.1 ± 36.8

395.6 ±20.9

Weight changes

 

 

Days 6-10

16.8 ± 7.0

22.5 ± 5.4

Days 10-15

33.4 ± 8.2

29.6 ± 6.4

Days 6-15

50.1 ± 11.3

52.2 ± 6.7

Group a: treated with 25 mg/kg bw

Group b: treated with 25 mg/kg bw, but erroneously once with 150 mg/kg bw

Food consumption:

Food consumption did not differ significantly between all groups during the evaluated intervals.

Clinical signs:

No treatment-related clinical signs were noted during the course of the study. The only clinical findings were alopecia in one animal of mid dose group and conjunctivitis in one animal of high dose group both in observation period of days 6 -15 of gestation.

No females were found dead or had to be killed in extremis prior to caesarean section on day 20.

Pregnancy performance:

No significant differences in mating performance of treatment groups were found compared to control group. No females showed signs of abortion or premature delivery during the course of the study.

Necropsy findings:

No treatment-related findings were observed during necropsy of females.

Caesarean section:

The mean number of live fetuses (per dam) and the number of fetuses (per dam) in the left or right uterine horns did not differ significantly among groups.

No biological relevant differences were determined among groups concerning the number of fetuses with head or tail presentation. The sex ratio and distribution show intergroup variation, but no biologically relevant differences are evident.

Fetal weights:

Table 5. Fetal weights (mean value ± sd)

 

Fetuses

Group

Males

Females

Males + Females

0 mg/kg

3.94 ± 0.28

3.76 ± 0.25

3.84 ± 0.27

25 mg/kg

4.06 ± 0.29

3.86 ± 0.28

3.95 ± 0.25

150 mg/kg

3.88 ± 0.22

3.68 + 0.18

3.79 ± 0.20

1000 mg/kg

3.59x ± 0.33

3.37 ± 0.35

3.51x ± 0.27

MSB

0.9723

1.0801

0.8549

MSW

0.0775

0.0747

0.0632

F

12.55***

14.45***

13.52***

DF

3/93

3/92

3/93

P

0.000

0.000

0.000

x   = p 0.05   (Dunnett-test); *** = p 0.001 (F-test)

Placental weights:

Mean placental weights were comparable between control and the dose groups.

Dead fetuses and live births index:

There were no dead fetuses in any test group. Consequently the Live Births Index was 100% in all groups.

Number of runts and Runts Index:

Table 6.No. of runts, runts index

group

No. of runts

Runts Index [%]

0 mg/kg

0

0

25 mg/kg

1

0.3

150 mg/kg

1

0.3

1000 mg/kg

6

1.6

Number of Resorptions and resorption index:

Table 7.Number of resorptions, resorption index (mean value ± sd)

group

early resorptions

late resorptions

total resorptions

Resorption index [%]

0 mg/kg

0.7 ± 0.9

0.0 ± 0.0

0.7 ± 0.9

4.4 ± 5.7

25 mg/kg

0.8 ± 0.9

0.1 ± 0.3

0.9 ± 0.9

6.0 ± 5.7

150 mg/kg

0.7 ± 0.8

0.1 ± 0.3

0.8 ± 0.8

5.3 ± 5.0

1000 mg/kg

1.3 ± 1.4

0.4 ± 1.1

1.7x ± 2.0

10.2 ± 12.0

x = p 0.05 (U-test)

Table 8. Relative frequency of females with resorptions (%)

group

0 mg/kg

25 mg/kg

150 mg/kg

1000 mg/kg

Without resorptions

54

36

42

25

With 1 resorption

25

44

33

33

With 2 or more resorptions

21

20

25

42

Pre-implantation loss index:

The pre-implantation loss indices did not differ significantly between the groups.


Post-implantation Loss Index:

Table 9.Post-implantation Loss Index (mean value ± sd)

group

Post-implantation Loss Index [%]

0 mg/kg

4.4 ± 5.7

25 mg/kg

6.0 ± 5.7

150 mg/kg

5.3 ± 5.0

1000 mg/kg

10.2 ± 12.0

Implantations:

Mean numbers of implantations showed no significant differences between groups and no biologically relevant differences were found in the left/right intrauterine distribution.

Corpora lutea:

Significantly increased number of corpora lutea at 1000 mg/kg bw. Accordingly, the number of copora lutea in each ovary was also increased. This finding is considered to be incidental, since the treatment commenced only on day 6 of gestation, when corpora lutea had already developed (table 10).

Table 10. No. of Corpora Lutea (Analysis of variance, mean values, standard deviations)

group

left

right

total

0 mg/kg

8.4 ± 1.9

9.0 ± 2.2

17.4 ± 2.1

25 mg/kg

8.5 ± 1.8

9.1 ± 2.1

17.6 ± 2.3

150 mg/kg

8.7 ± 1.8

9.1 ± 2.9

17.8 ± 2.9

1000 mg/kg

9.6 ± 2.6

10.2 ± 2.4

19.8x ± 2.8

MSB

7.6149

7.8864

30.8978

MSW

4.1831

5.8540

6.4009

F

1.82

1.35

4.83**

DF

3/93

3/93

3/93

P

0.147

0.263

0.004

x = p 0.05 (Dunnet-test); ** = p 0.01 (F-test)

External examination:

One incidental finding was observed in the control group. In the high dose group the hindlimbs appeared slightly crooked in two fetuses. Since no bent bones of the hindlimbs were found and only two fetuses were affected, this finding is considered to be without toxicological importance.

Visceral examination:

Slight to moderate hydrocephalus was observed in two fetuses of the high dose group in two different litters. A clear treatment relation cannot be considered for this malformation since this finding occurred in control fetuses of the historical studies even with a slightly higher incidence.

The finding of diaphragmatic hernia found in three fetuses of two litters of the high dose group was also found in literature studies with a spontaneous occurrence of 2.7 % in albino rats [Andersen d. H. (1949) Effect of diet during pregnancy upon the incidence of congenital hereditary diaphragmatic hernia in the rat. Am. J. Pathol. 25, 163 -185] and up to 0.9 % of fetuses of vehicle treated groups and in up to 5.6 % of litters [Charles River Laboratories (1988) Embryo and fetal developmental toxicity (teratology) control data in the Charles River Crl:CD(R) BR rat. Charles River Laboratories, ]. Andersen (1949) denominated this kind of abnormality as a right incomplete diaphragmatic hernia and stated, that this lesion appeared to have no effect on the health or growth of the affected fetuses. Diaphragmatic henia with protusion of a part of the liver can be considered as a result of delayed development of pleuraoperitoneal membranes. This delay of development may be associated with the general growth retardation which was seen in fetuses of high dose group females.

A slight tendency of increased occurrence of dilated ureters may be stated with respect to affected fetuses in the dose groups. However, the relative frequency of affected litters was very similar in control, low and mid dose group. The slight increase of dilated ureters in the high dose group may be associated with the general growth retardation in fetuses of the high dose group.

Dilation of renal pelvis was noted in fetuses of all test groups with a similar incidence and no dose relationship.

All other abnormalities of soft tissues were minor findings and were noted in single fetuses only. Thus they are considered to be without toxicological relevance.

Skeletal examination:

One malformed fetus was found in the high dose group showing as major finding a kyphosis of the thoracic vertebral column. Since it occurred only once and scoliosis, which denotes a similar abnormality of the vertebral column, was found even in historical control fetuses, this finding is considered to be incidental.

The relative frequency of non-ossified sternebrae was very similar between all test groups with respect to both affected fetuses and litters. The incidence of other skeletal abnormalities provided no evidence of a treatment effect.

Table 11. Skeletal examination: Number of ossification centers (Relative frequencies [%] of affected fetuses and litters)

Group

0 mg/kg

25 mg/kg

150 mg/kg

1000 mg/kg

No. of examined fetuses/litters

Fetuses / litters

188 / 24

189 / 24

180 / 24

191 / 24

Number of ossification centers

Sternum

6

Fetuses/litters

73 / 96

77 / 100

75 / 96

68 / 96

5

22 / 75

16 / 64

23 / 58

25 / 88

4

4 / 21

6 / 32

2 / 13

5 / 29

4

1 / 4

1 / 4

1 / 4

3 / 21

Metacarpals

4

Fetuses / litters

82 / 100

88 / 100

84 / 100

66 / 100

3

18 / 58

12 / 52

16 / 67

34 / 88

3

1 / 4

0 / 0

0 / 0

0 / 0

Metatarsals

¿ 4

Fetuses /litters

100 / 100

100 / 100

99 / 100

100 / 100

3

0 / 0

0 / 0

1 / 4

0 / 0

3

0 / 0

0 / 0

0 / 0

0 / 0

Table 12: Skeletal examination: (Selected) skeletal abnormalities

 

group

0 mg/kg

25 mg/kg

150 mg/kg

1000 mg/kg

 

 

fetuses (%)

litters (%)

fetuses (%)

litters (%)

fetuses (%)

litters (%)

fetuses (%)

litters (%)

No. of examined fetuses/litters

188 (100)

24 (100)

189 (100)

25 (100)

180 (100)

24 (100)

191 (100)

24 (100)

Sternum (ossification centers)

150 (80)

24 (100)

150 (79)

25 (100)

128 (71)

24 (100)

158 (83)

24 (100)

 

irregular shape

34 (18)

20 (83)

54 (29)

20 (80)

39 (22)

17 (71)

58 (30)

22 (92)

 

cleaved

1 (1)

1 (4)

1 (1)

1 (4)

1 (1)

1 (4)

5 (3)

5 (21)

 

rudimentary

48 (26)

20 (83)

30 (16)

15 (60)

28 (16)

15 (63)

51 (27)

19 (79)

 

incomplete ossification

93 (49)

24 (100)

98 (52)

25 (100)

94 (52)

23 (96)

120 (63)

24 (100)

 

not ossified

52 (28)

20 (83)

43 (23)

18 (72)

45 (25)

15 (63)

58 (30)

20 (83)

Vetebral column

27 (14)

11 (46)

26 (14)

12 (48)

45 (25)

18 (75)

81 (42)

23 (96)

 

Thoracic Vertebrae

27 (14)

11 (46)

26 (14)

12 (48)

45 (25)

18 (75)

78 (41)

21 (88)

 

kyphosis

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

1 (1)

1 (4)

 

vertebral bodies

27 (14)

11 (46)

26 (14)

12 (48)

45 (25)

18 (75)

77 (40)

21 (88)

 

cleaved

0 (0)

0 (0)

0 (0)

0 (0)

4 (2)

3 (13)

16 (8)

9 (38)

 

dumpbell shaped

27 (14)

11 (46)

26 (14)

12 (48)

44 (24)

18 (75)

74 (39)

21 (88)

Coccygeal Vertebrae

0 (0)

0 (0)

1 (1)

1 (4)

1 (1)

1 (4)

5 (3)

4 (17)

 

not ossified

0 (0)

0 (0)

1 (1)

1 (4)

1 (1)

1 (4)

5 (3)

4 (17)

Variation indices:

Variation indices did not show any biologically significant differences.

Anomaly index and malformation index:

Table 13.Anomaly index and malformation index

group

0 mg/kg

25 mg/kg

150 mg/kg

1000 mg/kg

Number of anomalies skeletal

29

35

45

81

Anomaly Index skeletal

15.4

18.5

25.0

42.4

Litters affected skeletal

11

14

18

23

%

45.8

56

75

95.8

Number of malformed fetuses

0

0

0

6

malformaion Index

0.0

0.0

0.0

1.6

Litters affected

0

0

0

4

%

0.0

0.0

0.0

16.7

Conclusions:
The test substance caused a clear maternal toxic effect at 1000 mg/kg bw/d shown as a dose-dependent decreased body weight gain. Body weight effects at 150 mg/kg were only transitory and slight.
Embryotoxic effects were found as distinct growth retardation of fetuses and to an increased number of resorptions at 1000 mg/kg bw/d. The growth retardation is considered to be the cause of many of the findings seen during fetal examination, e. g. diaphragmatic hernia, increase of dilated ureters, skeletal findings. At doses of 150 mg/kg bw/d only a very slight occurrence of delayed ossification was found.

Under the conditions of this study, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity was considered to be 150 mg/kg bw/d. The No Observed Adverse Effect Level (NOAEL) for developmental toxicity was considered to be 150 mg/kg bw/d, since at this dose only a very slight occurrence of delayed ossification was observed, accompanied by a distinct maternal toxicity.
Executive summary:

The test material was administered to pregnant Wistar rats via oral gavage from day 6 to 15. At doses of 25 and 150 mg/kg bw/d no maternal toxicity was observed. At a dose of 1000 mg/kg/d clear maternal toxicity, such as reduced body weight gain was observed.

At 25 mg/kg bw/d no effects on offspring was observed. At a dose of 150 mg/kg bw/d only a very slight delay of ossificaiton occurred. Treatment of the females with the high dose (1000 mg/kg bw/d) led to a distinct growth retardation of fetuses and an increased resorption. The growth retardation can be seen as cause of the various findings observed during fetal examination.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
some information such as the purity or batch number of the test substance or concerning the exposure conditions are lacking.
GLP compliance:
not specified
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: no data
- Age at study initiation: 14 weeks for males and 12-13 weeks for females
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: individual aluminium cages for the gravid females
- Diet (e.g. ad libitum): solid diet (aliment MF, oriental yeast Co., Ltd.)
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period:


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 +/- 1
- Humidity (%): 55 +/- 5
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 (6:00 to 18:00)
Route of administration:
oral: gavage
Vehicle:
other: Sesame oil (japanese pharmacopea)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: no data

VEHICLE
- Justification for use and choice of vehicle (if other than water): dibenzyltoluene is not soluble in water
- Concentration in vehicle: 50%
- Amount of vehicle (if gavage): 2, 06 and 0.2 ml/kg, respectively for the dose-levels of 1000, 300 and 100 mg/kg/d
- Lot/batch no. (if required): no data
- Purity: no data
Details on analytical verification of doses or concentrations:
No data
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: no data
- Length of cohabitation: one night
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
from day 7 to day 17 of gestation
Frequency of treatment:
once a day
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
20 dams
Control animals:
other: sesame oil
Details on study design:
- Dose selection rationale: The high-dose level was fixed according to preliminary assays showing a decrease in the body weight gain of gravid females given 1000 mg/kg/d of dibenzyltoluene.

Maternal examinations:
CLINICAL OBSERVATION: daily

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION : Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/rat/day

WATER CONSUMPTION: No data

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on 21 day of gestation
- Organs examined: no data
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of : Yes
- Number of dead and liv conceptuses : Yes
Fetal examinations:
- External examinations: Yes (third per litter )
- Soft tissue examinations: Yes (third per litter ) after fixation for 2 weeks in Bouin's mixture according to the wilson's method
- Skeletal examinations: Yes (third per litter )
- Head examinations: Yes (third per litter )
- fetal weight: Yes
Statistics:
- Implantation rate: Chi-square
- Weight of dams, food consumption, number of corpora lutea, number of implants, number of fetuses, fetal body weight: Student's t test (if homogeneous variance), Aspin-Welch t test (in other cases)
- Fetal mortality, sex ratio, abnormalities frequency: Wilcoxon test.
Indices:
No data
Historical control data:
No data
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL FINDINGS:
Piloerection was observed in the highest dose group (1000 mg/kg/day).

MORTALITY:
Maternal mortality was not reported.

BODY WEIGHT:
A slight decrease in body weight gain was noted in dams exposed to 1000 mg/kg/day when compared to that of control.

FOOD CONSUMPTION:
A visible decrease in food intake was observed at 1000 mg/kg/day but no dose-related effect was found.

NECROPSY:
- Fertility parameters:
. All dams had live fetuses.
. A slight but statistically significant decrease in the number of corpora lutea was observed at 1000 mg/kg/day (274 corpora lutea in 20 pregnant females) when compared to control (299 corpora lutea) however since dibenzyltoluene was administered after the implantation should be performed (day 6), this effect was not considered to be treatment-related .
. Number of implants were unaffected by treatment and implant ratio was similar in all groups.

Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No teratogenic effects were found.
- Mortality:
. Fetal mortality was similar in treated and control groups.
- Litters examination:
. Litter sizes in all groups were similar.
. No significant difference in sex ratio was found.
. Pups body weight was nonetheless statistically reduced in the highest dose group when compared to control (3.8 vs 4.1 g in male, 3.6 vs 3.9 g in females).
. No toxicologically significant malformations were observed.
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
Under these experimental conditions, the No-Observed-Adverse-Effect-Level (NOAEL) was 300 mg/kg/d for maternal and foetal toxicity.
Executive summary:

The potential of Dibenzylbenzene, ar-methyl derivative to induce developmental toxicity after maternal exposure during the critical period of organogenesis was evaluated in rat according to a study design comparable to OECD Guideline N° 414.

Dibenzylbenzene, ar-methyl derivative was administered orally by gavage to three groups of 20 bred female Sprague-Dawley rats once daily from gestation days 7 through 17. Dosage levels were 0, 100, 300 and 1000 mg/kg/d.

Animals were observed daily for mortality and morbidity. Clinical observations, body weights and food consumption were recorded at appropriate intervals.

On gestation day 20, a hysterectomy was performed on each female. The uteri, placenta and ovaries were examined, and the number of foetuses, resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded. The foetuses were weighted, sexed and examined for external, visceral and skeletal malformations and developmental variations.

All animals survived to the scheduled necropsy. Piloerection was observed in females given 1000 mg/kg/d as well as a slight decrease in body weight gain and food consumption.

The slight increase in the resorption of corpora lutea noted in the highest dose group was not considered as relevant since the administration of Dibenzylbenzene, ar-methyl derivative occurred after the implantation. The survival of the pups was unaffected by Dibenzylbenzene, ar-methyl derivative administration at all dose levels. However, pups body weight was reduced in the highest dose group when compared to controls but this effect was related to the maternal toxicity observed. Foetal external, soft tissue and skeletal malformations observed in control and treated pups were considered to be spontaneous in origin. The developmental variations expressed in the treated groups were generally similar to those present in the control group or occurred in a manner that was not dose related.

Under these experimental conditions, the No-Observed-Adverse-Effect-Level (NOAEL) was 300 mg/kg/d for maternal toxicityand the No-Observed-Adverse-Effect-Level (NOAEL) was 1000 mg/kg/d for fetal toxicity.

Effect on developmental toxicity: via oral route
Dose descriptor:
NOAEL
150 mg/kg bw/day
Additional information

The effects of Dibenzylbenzene, ar-methyl derivative on the development was evaluated in two separate developmental toxicity study in rats. No treatment related specific effects on the development of fetuses were observed. Findings observed in the studies were either slight and/or can be related to the maternal toxicity caused by treatment. In one study a more distinct toxicity to maternal animals was observed. In that study the test material was administered to pregnant Wistar rats via oral gavage in doses of 25, 150 or 1000 mg/kg bw/d. At doses of 25 mg/kg bw/d no maternal toxicity was observed. At doses of 150 and 1000 mg/kg/d clear maternal toxicity, such as reduced body weight gain was observed.

At 25 mg/kg bw/d no effects on offspring was observed. At a dose of 150 mg/kg bw/d only a very slight delay of ossification occurred. Treatment of the females with the high dose (1000 mg/kg bw/d) led to a distinct growth retardation of fetuses and an increased resorption. The growth retardation can be seen as cause of the various findings observed during fetal examination.

Justification for classification or non-classification

No classification for reproductive or developmental toxicity is indicated according to the general classification and labeling requirements for dangerous substances and preparations (67/544 EEC) or the classification, labeling and packaging (CLP) regulation (EC 1272/2008).