Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Screening for productive/developmental toxicity study(OECD 412,GLP):

NOAEL for male rats: 5 mg/kg bw/day
NOAEL for female rats: 5 mg/kg bw/day
NOAEL for reproductive performance of the male rats: 5 mg/kg bw/day
NOAEL for reproductive performance of the female rats: 5 mg/kg bw/day
NOAEL for F1 Offspring: 5 mg/kg bw/day O R

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-06-07 to 2014-01-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Hsd.Brl.Han:Wistar rat

Details on species / strain selection:

The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to a wide range of experience with this strain in reproduction toxicity studies and well known fertility parameters.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test Animals
Species and strain: Hsd.Brl.Han:Wistar rat
Source: Toxi-Coop Zrt. Cserkesz u. 90. H-1103 Budapest Hungary
Hygienic level at arrival: SPF
Hygienic level during the study:Good conventional
Number of animals: 48 males, 48 females (12 not treated male animals for a second mating in 40 and 15 mg/kg bw/day groups (not treated satellite group))
Sex: Males and nulliparous, non-pregnant females
Age of animals at start: Male animals: 80 – 85 days old Female animals: 80 – 85 days old
(Male animals of the second mating were 68-76 days old at the commencement of pairing)
Body weight range at start: Male animals: 314 – 367 g Female animals: 180 – 218 g The weight variation in animals involved at the starting point of the study did not exceed ± 20 % of the mean group weight of each sex.
Acclimatization time: 20 days (6 days for male animals of the second mating)
Animal Husbandry
Animal health: Only healthy animals were used for the test. The breeder certified their health status. (See Appendix XIV)
Room number: 4/A
Housing: Before mating: 2 animals of the same sex/cage
Mating: 1 male and 1 female/cage Pregnant females were housed individually. Males after mating: 2 animals/cage
Cage type: Type III polypropylene/polycarbonate Size: 22 x 32 x 19 cm (width x length x height)
Bedding: Lignocel Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (D-73494 Rosenberg, Holzmühle 1, Germany). (See Appendix XVII for Quality certificate of bedding material).
The bedding was suitable as nesting material. Cages and bedding material were changed twice a week.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 - 70 %
Ventilation: 10-15 air exchanges/hour by central air-condition system.
The temperature and relative humidity was checked and recorded once daily during the study.

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % Methylcellulose

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:


VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is not soluble in water; therefore 0.5 % aqueous methylcellulose was used for preparing formulations appropriate for oral administration. 0.5 % aqueous methylcellulose was a suitable vehicle to facilitate formulation analysis for the test item.

The test item was formulated in the vehicle in concentrations of 8, 3 and 1 mg/mL. Formulations were prepared in the formulation laboratory of Test Facility beforehand and not longer than three days before the application.
Analysis of formulations was performed in the Analytical Laboratory of Test Facility. Five samples were taken (10 mL, each, from different places) from each concentration (Groups 2, 3 and 4) and measured. Similarly, five samples were taken from the control solution (10 mL, each, from different places, group 1) and analyzed.

Details on mating procedure:

Mating began 2 weeks after the initiation of treatment with one female and one male of the same dose group (1:1 mating) placed in a single cage. Females remained with the same male until copulation occurred. 10 female animals of the 40 mg/kg bw/day and two ones in the 15 mg/kg bw/day group showed no sign of copulation after 14 days mating. Therefore pairs were changed within the dose group i.e. females were re-mated with other not mated males of the same dose group between Days 28 and Day 34. Due to the failed pairing, females in the 15 mg/kg bw/day group were re-mated with proven males between Days 35 and 41 to achieve 8 pregnant females.
A second mating of the parental females in the high and mid dose groups was conducted with untreated males from Day 35 to Day 48 and from Day 42 to 48, respectively to clarify and for further characterization of effects on fertility observed in the first mating. None of the high dose animals became pregnant after the overall mating period of 35 days.
Each morning a vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (day 0 of pregnancy as defined by OECD 421). Sperm positive females were caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration of the test item in the dosing formulations varied from 108 % to 119 % of nominal concentrations and samples proved to be homogenous at both analytical occasions, thereby confirming proper dosing.

Duration of treatment / exposure:

The test item was administered in a single dose by oral gavage on a 7 days/week basis, every day at a similar time. Control animals were treated concurrently with the vehicle only. Animals were not treated on the day of gross pathology.
Dosing of both sexes began after acclimatization and was continued up to and including the day before the necropsy.
The mating phase started after 14-days of pre-mating. Rats of this strain have already reached full sexual maturity at the age of 12 weeks. Male animals were dosed for 42 days and then they were subjected to necropsy one day after the last treatment (Day 42).

Frequency of treatment:

everyday

Details on study schedule:

The test item was administered in a single dose by oral gavage on a 7 days/week basis, every day at a similar time. Control animals were treated concurrently with the vehicle only. Animals were not treated on the day of gross pathology.
Dosing of both sexes began after acclimatization and was continued up to and including the day before the necropsy.
The mating phase started after 14-days of pre-mating. Rats of this strain have already reached full sexual maturity at the age of 12 weeks. Male animals were dosed for 42 days and then they were subjected to necropsy one day after the last treatment (Day 42).

Female animals were dosed for 14 days pre-mating, during mating period, through gestation and up to lactation days 3 – 6 (altogether for 41 – 47 days, depending on date of mating). The day of delivery (i.e. when parturition was complete) was defined as day 0 post-partum or lactation day 0.

Non-pregnant and not mated female animals in the control, low or mid dose groups were treated up to and including the day before necropsy (altogether for 42 – 50 days). Female animals subjected to the second mating (i.e. 10 animals of the high dose group and 2 of the mid dose group) were treated for 50 – 72 days.
Control animals were handled in an identical manner to the test groups receiving only the vehicle (5 mL/kg bw/day).

Dose / conc.:

40 mg/kg bw/day (nominal)

Dose / conc.:

15 mg/kg bw/day (nominal)

Dose / conc.:

5 mg/kg bw/day (nominal)

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control group

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose setting was based on findings obtained in a previous 14-day oral gavage dose range finding study with Reaction product of 2-Hydroxybenzoic Acid, Styrene and Oxozinc in rats (Toxi-Coop study no. 722.400.4163) and in agreement with the Sponsor. The high dose was chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect.

Parental animals: Observations and examinations:

Clinical Examination：
Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).
General clinical observations were made once a day, after treatment at approximately the same time, considering the peak period of anticipated effects after dosing.
More detailed examinations were made weekly at the times of weekly weighing prior to and during the mating until necropsy. Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling.

Weight Assessment：
All parental animals were weighed with an accuracy of 1 g.
Parental males were weighed on the first day of dosing (Day 0), weekly thereafter and on the day of necropsy.
Parental females were weighed on the first day of dosing (Day 0) then weekly, on gestation days 0, 7, 14 and 21 and on days 0 (within 24 hours after parturition) and 4 post-partum. Body weight of the female animals were additionally determined on gestational days 10 and 17 in order to give accurate treatment volumes, but these data were not evaluated and reported.
The body weight of animals in the control and all dosed groups were determined daily from day 7 up to normalization of the body weight gain (Day 14) and occasionally twice a week thereafter. The body weight significantly decreased in male and female animals of 40 mg/kg bw/day and of 15 mg/kg bw/day during the first week (between Days 0 and 7). In order to give accurate treatment volumes and handling of all animals in the same way, the body weight was determined daily and the treatment volume was adjusted on the basis of the most recent individual body weight measurement daily.
The body weight of female animals was measured and recorded weekly during the second pairing but these data were not reported.

Food Consumption：
The food consumption was determined with a precision of 1 g weekly by re-weighing the non-consumed diet during the treatment period (pre-mating, gestation days 0, 7, 14 and 21, lactation days 0 and 4) except during mating phase.

Sperm parameters (parental animals):

percentage of sperm positive,but non-pregnant female

Litter observations:

− Litter weight on postnatal days 0 and 4,
− Mean body weight gain per litter between postnatal days 0-4
− Number of live births per litter, and number of viable pups per litter on postnatal days 0 and 4
− Survival Index of pups on postnatal day 4
− Sex ratio % (on postnatal days 0 and 4).

Postmortem examinations (parental animals):

Gross necropsy was performed on each animal. One day after the last treatment, animals were euthanized by exsanguination after verification of the deep narcosis by Isofluran CP (details are presented in "Details of Other Materials").
After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed. Any abnormality was recorded including details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded.
The testes, epididymides and brain of all male adult animals were weighed.
The uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, ovaries, pituitary and all organs showing macroscopic lesions of all adult animals were preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution.

Postmortem examinations (offspring):

Offspring euthanized on postnatal day 4 were carefully examined for gross abnormalities.

Statistics:

The statistical evaluation of appropriate data (marked †above) was performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test.
Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, then Duncan Multiple Range test was used to assess the significance of inter-group differences. Getting significant result at Bartlett’s test the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed, if feasible.
The frequency of clinical signs, pathology and histopathology findings were calculated.
Results were evaluated in comparison with values of control group (i.e. control value). Parameters indicated with statistical significances were listed as deviations from control value in paragraph “Results”.

Reproductive indices:

Mating / Copulatory Index (Measure of animals’ ability to mate)
Fertility Index (Measure of male’s ability to produce sperm that can fertilize eggs and Measure of female’s ability to become pregnant)
Gestation Index (Measure of pregnancy that provides at least one live pup)

Offspring viability indices:

Pup Mortality and Sex Ratio Indices

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test item related clinical signs were observed in male and female animals of 40 mg/kg bw/day groups (8/12 and 12/12, respectively) and in female animals of 15 mg/kg bw/day (7/12).
Pale (3/12 male, 8/12 female) or cold (7/12 male, 12/12 female) limbs, decreased activity (2/12 male, 1/12 female) and piloerection (1/12 male and 4/12 female) were detected in animals administered with 40 mg/kg bw/day dose. Decreased activity (1/12 male), pale and cold limbs (3/12 female and 7/12 female, respectively) were also observed in animals of 15 mg/kg bw/day group. Decreased activity of one single male animal of 15 mg/kg bw/day group was considered to be individual signs as no other sings were detected and there were no other animals with similar findings.
Dermal alterations such as alopecia or scab were observed in single animals of high, middle and control groups as follows: alopecia on the neck in in 2/12 female animals at 15 mg/kg bw/day and on the limbs in 1/12 control male animal, on the abdomen in 1/11 dam of control group; scab on the back in 1/12 male animals at 40 mg/kg bw/day, on the right side of the body in 1/12 male and on the lower part of the neck 1/12 female at 15 mg/kg bw/day; on the right fore limb in 1/12 male control animal. Alopecia and scab on the skin are common dermal findings in this strain of experimental rats and were observed in control and treated animals with similar incidence. Therefore, these had no toxicological meaning in this study.

Mortality:

no mortality observed

Description (incidence):

There was no mortality during the course of the study (control, 40, 15 or 5 mg/kg bw/day groups).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight development was reduced in male and female animals of 40, 15 and 5 mg/kg bw/day groups with respect to their controls during the entire study.
Significantly less summarized mean body weight gain (between Days 0 and 41) was detected in the male animals of 40, 15 and 5 mg/kg bw/day with respect to the control. The weekly mean body weight gain of male animals was reduced comparing to the control group on each week (except on week 3) in the 40 mg/kg bw/day group with statistical significances. Statistically significant reduced mean body weight gain was also observed on weeks 1, 2 and 4 in the 15 and 5 mg/kg bw/day groups and on week 6 in the 15 mg/kg bw/day group. The reduced body weight gain resulted in a less mean body weight in all male groups with statistical significances from Day 7 (40 and 15 mg/kg bw/day) or from Day 13 (5 mg/kg bw/day) during the entire study.
The mean total body weight gain (between Days 0 and 13) was also significantly less than in the control group in female animals administered with 40, 15 or 5 mg/kg bw/day groups. The significantly less body weight gain on week 1 resulted in reduced mean body weight in all female treated groups on Days 7 and 13 with respect to the control.
The body weight development was also depressed in dams administered with 15 mg/kg bw/day during the gestation and lactation periods. Significantly less mean body weight gain was detected in the mid dose group during the entire gestation and lactation periods.
A slight but statistically significantly reduced mean body weight gain (gestation week 1) and mean body weight of dams was observed in the 5 mg/kg bw/day group (gestation days 7 and 14, and lactation day 4) with respect to the control group.
The differences between the control and 5 mg/kg bw/day groups (male and female) were small (<10 %) therefore were not considered to be of toxicological relevance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Test item related effect on the mean daily food consumption was observed in male and female animals in 40, 15 and 5 mg/kg bw/day groups.
The mean daily food intake was statistically significantly less than in the control group in full correspondence with the body weight development in male and female animals of all groups (40, 15 and 5 mg/kg bw/day) during the pre-mating period, in male animals of 40 and 15 mg/kg bw/day between Days 34 and 41, as well as in dams of 15 mg/kg bw/day during the gestation and lactation period.
Similarly, the mean daily food consumption was slightly less comparing to control group in male animals at 5 mg/kg bw/day between Days 27 and 34 and in female animals at 5 mg/kg bw/day between gestation days 7 and 14. The differences between the control and 5 mg/kg bw/day groups (male and female) were small (<14 %) therefore were not considered to be of toxicological relevance.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histological examination revealed sings of decreased secretion in the tubules (less amount of secretion and narrow diameter of tubules) of the seminal vesicles with coagulating gland and prostate in male animals of 40 mg/kg bw/day (6/12).
In five of these animals (5/12), decreased intensity of spermatogenesis was observed in a part (40-50 %) of seminiferous tubules in the testes, and lack of mature spermatozoa was also detected in the ductuli of epididymides of these animals. In the affected tubules of the testes, the lack of mature spermatozoa and spermatids was detectable. The Sertoli-cells, the spermatogonia and spermatocytes were intact. In the other 50-60% of the seminiferous tubules, the histological picture of active spermatogenesis was seen.
These findings were not accompanied with inflammation, degeneration or necrosis in the affected organs and seemed to be functional ones and reversible in character. The number and cytomorphology of interstitial testicular cells were normal and similar to the control male animals.
In the male animals of 15 and 5 mg/kg bw/day, group, the investigated organs of reproductive system (testes, epididymides) were histologically normal and characteristic for the sexually mature organism in all cases of control and treated groups. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa), representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphology in the testes of the investigated animals. Histologically, epididymides were normal in all cases as well.
Involution of part of corpora lutea and part of secondary and tertiary follicles was observed in the ovaries of all female animals belonging to the high dose group (12/12). Similarly, involution of part of corpora lutea (5/12) and part of secondary and tertiary follicles (11/12) was observed in the ovaries of female animals of the 15 mg/kg bw/day group. The complete lack of corpora lutea was detected in several animals (10/12) in the high dose group. The primary follicles, and the oocytes surrounded by stratified epithelium in the primary follicles, and the ovarian interstitial endocrine cells were intact in all the affected animals. The other proportion of secondary and tertiary follicles was also normal in the ovaries of animals in the high and middle dose groups. Similarly, the other proportion of corpora lutea in the middle dose group was normal as well.
These findings were not accompanied with inflammation or necrosis in the affected genital organs and were to be as functional and reversible in character.
In the female animals of 5 mg/kg bw/day group including non-pregnant animal, the ovaries had a normal structure characteristic for the species, age and phase of sexual cycle. The cortex contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes and ovulation. The epithelial capsule and ovarian stroma were normal in all cases as well.
In one control male animal (1/12) one side pyelectasia was observed. This finding (without degenerative, inflammatory or fibrotic lesion) is an individual disorder without toxicological significance.

Histopathological findings: neoplastic:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

Test item related changes were found in the reproductive performance of male and female animals administered with 40 mg/kg bw/day dose of the test item.
There was no fertile mating in 40 mg/kg bw/day group, i.e. the fertility index was zero both for male and female animals. The number of not mated animals (male and female 10/12) was high, the number of mated animals i.e. the copulatory index was significantly less than in the control group (male and female). The pre-coital interval was also significantly elongated at 40 mg/kg bw/day dose with respect to control and historical control.
Statistical significances were noted for the higher percentage of not mated animals and less percentage of mated or sperm positive animals (i.e. copulatory index) in the 15 mg/kg bw/day group (male and female). The percentage of fertile males and pregnant females, as well as the fertility indices (male and female) were also less, the percentage of non-pregnant females was higher in 15 mg/kg bw/day treated group with respect to their control. However, all these statistically significant differences with respect to the appropriate control were judged to be toxicologically not relevant as all values were similar to the historical control data.
There were no test item related differences between the control and low dose (5 mg/kg bw/day) treated animals in the examined parameters of reproductive performance.
The second mating of female animals with not treated males did not result in fertile mating. Therefore it could be concluded, that the reproductive performance of female animals of the 40 mg/kg bw/day group was also affected by the test item.

Dose descriptor:

NOAEL

Effect level:

5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

gross pathology

histopathology: non-neoplastic

reproductive performance

Critical effects observed:

yes

Lowest effective dose / conc.:

40 mg/kg bw/day (nominal)

System:

female reproductive system

Organ:

other: genital organ

Treatment related:

yes

Dose response relationship:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

40 mg/kg bw/day (nominal)

System:

male reproductive system

Organ:

other: male genital organs and accessory sex organs

Treatment related:

yes

Dose response relationship:

yes

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Test item related toxic clinical signs did not appear in the offspring of 15 or 5 mg/kg bw/day groups.
The percentage of cold offspring was slightly higher in the 15 and 5 mg/kg bw/day groups with respect to the control. However this sign was only transient and was observed on postnatal day 0. Therefore it was not considered to be of toxicological relevance.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There was no test item effect on extra uterine mortality of offspring (15 and 5 mg/kg bw/day).
All offspring of 15 and 5 mg/kg bw/day group survived up to postnatal day 4. Statistical significance was noted for the less litter mean of offspring number on postnatal day 4. However this difference with respect to control was also present on postnatal day 0.
One offspring of one control dam was missing on postpartal day 3 probably due to cannibalism.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean litter weight and weight gain were slightly less in offspring of 15 mg/kg bw/day group with respect to control due to the less number of offspring. The mean offspring weight was similar to the control value on postnatal days 0 and 4. However the mean weight gain of offspring was slightly less with respect to control in the 15 and 5 mg/kg bw/day groups between postnatal days 0 and 4.

Gross pathological findings:

not examined

Description (incidence and severity):

There were no dead or stillborn offspring, therefore no necropsy was conducted.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

other: Sex distribution

Critical effects observed:

no

Reproductive effects observed:

yes

Lowest effective dose / conc.:

15 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects in the absence of other toxic effects

Dose response relationship:

yes

Conclusions:

Under the conditions of the present study Reaction Product of 2-Hydroxybenzoic Acid, Styrene and Oxozinc caused clinical signs, reduced body weight development and food consumption (male and female), macroscopic and weight changes accompanied by histological alterations in male genital organs and accessory sex organs, and histological alterations in female genital organ in Hsd.Brl.Han: Wistar rats after repeated dose oral administration at 40 mg/kg bw/day. The high dose caused infertility of female animals and probably also of male animals.
At 15 mg/kg bw/day, test item induced clinical signs in female animals, reduced body weight and body weight gain and reduced food consumption in male and female animals. Dam’s delivery data was slightly affected by the test item and offspring’s body weight development was slightly depressed between postnatal days 0 and 4.
In the 5 mg/kg bw/day group, the slight changes in the body weight and food consumption were judged to be toxicologically not relevant.
Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:
NOAEL for male rats: 5 mg/kg bw/day
NOAEL for female rats: 5 mg/kg bw/day
NOAEL for reproductive performance of the male rats: 5 mg/kg bw/day
NOAEL for reproductive performance of the female rats: 5 mg/kg bw/day
NOAEL for F1 Offspring: 5 mg/kg bw/day

Executive summary:

A reproduction/developmental toxicity screening test was performed to obtain initial information on the possible effects of the test item Reaction Product of 2-Hydroxybenzoic Acid, Styrene and Oxozinc on reproduction and development when administered orally (by gavage) to rats at repeated doses of 40, 15 or 5 mg/kg bw/day compared to control animals according to OECD TG 421 under GLP compliance.

Results

Mortality
There was no mortality in any group (control, 40, 15 or 5 mg/kg bw/day).

Clinical observation
Test item related clinical signs (pale or cold limbs, decreased activity and piloerection) were observed in male and female animals of 40 mg/kg bw/day group. Pale and cold limbs were also detected in female animals of 15 mg/kg bw/day group.
At the detailed weekly observations, the above listed, test item related signs appeared in male (pale or cold limbs, decreased activity and piloerection) and female (pale and cold limbs and piloerection) animals of 40 mg/kg bw/day group. Pale and cold limbs were also detected in female animals of 15 mg/kg bw/day group in the course of weekly clinical observation indicating the cumulative effect of the test item.
Body weight and body weight gain
The body weight development was reduced in male and female animals of 40, 15 and 5 mg/kg bw/day groups with respect to their controls during the entire study. However the changes in 5 mg/kg bw/day group were not considered to be of toxicologically relevant due to the low degree.
Food consumption
Test item related effects on the mean daily food consumption were observed in male and female animals in 40, 15 and 5 mg/kg bw/day groups. However the changes in 5 mg/kg bw/day group were not considered to be of toxicologically relevant due to the low degree.
Reproduction
Test item related changes were detected in the reproductive performance of male and female animals administered with 40 mg/kg bw/day dose of the test item. The high dose caused infertility of female animals and probably also of male animals.
The delivery data of female animals administered with 15 mg/kg bw/day was influenced by the test item as the mean number of total births, live-borns, viable offspring and corpora lutea was significantly less and the mean post-implantation loss was higher in 15 mg/kg bw/day group with respect to the control.
Organ pathology
Smaller than normal testes, epididymides, seminal vesicles and prostate were noted for some male animals in the 40 mg/kg bw/day dose group supported by the results of organ weight evaluation (testes). Histological examination revealed decreased secretion in the tubules of the seminal vesicles with coagulating gland and prostate, decreased intensity of spermatogenesis in seminiferous tubules in the testes, and lack of mature spermatozoa in the ductuli of epididymides 40 mg/kg bw/day. In the testes, the lack of mature spermatozoa and spermatids was detectable.
Involution of corpora lutea and secondary and tertiary follicles was observed in the ovaries of all female animals belonging to the high dose group and in some animal of the 15 mg/kg bw/day group. The complete lack of corpora lutea was detected in several animals in the high dose group.

 

Offspring
There was no offspring in the 40 mg/kg bw/day group. A test item effect on the offspring development was observed in the less mean offspring weight gain in 15 mg/kg bw/day group between postnatal days 0 and 4.

 

Conclusion
Under the conditions of the present study Reaction Product of 2-Hydroxybenzoic Acid, Styrene and Oxozinc caused clinical signs, reduced body weight development and food consumption (male and female), macroscopic and weight changes accompanied by histological alterations in male genital organs and accessory sex organs, and histological alterations in female genital organ in Hsd.Brl.Han: Wistar rats after repeated dose oral administration at 40 mg/kg bw/day. The high dose caused infertility of female animals and probably also of male animals.
At 15 mg/kg bw/day, test item induced clinical signs in female animals, reduced body weight and body weight gain and reduced food consumption in male and female animals. Dam’s delivery data was slightly affected by the test item.
The offspring’s body weight development was slightly depressed between postnatal days 0 and 4.
In the 5 mg/kg bw/day group, the slight changes in the body weight and food consumption were judged to be toxicologically not relevant.
Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:
NOAEL for male rats: 5 mg/kg bw/day
NOAEL for female rats: 5 mg/kg bw/day
NOAEL for reproductive performance of the male rats: 5 mg/kg bw/day
NOAEL for reproductive performance of the female rats: 5 mg/kg bw/day
NOAEL for F1 Offspring: 5 mg/kg bw/day

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

5 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

High quality

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Toxicity to reproduction: other studies

Additional information

A reproduction/developmental toxicity screening test was performed to obtain initial information on the possible effects of the test item Reaction Product of 2-Hydroxybenzoic Acid, Styrene and Oxozinc on reproduction and development when administered orally (by gavage) to rats at repeated doses of 40, 15 or 5 mg/kg bw/day compared to control animals according to OECD TG 421 under GLP compliance.

Results

Mortality
There was no mortality in any group (control, 40, 15 or 5 mg/kg bw/day).

Clinical observation
Test item related clinical signs (pale or cold limbs, decreased activity and piloerection) were observed in male and female animals of 40 mg/kg bw/day group. Pale and cold limbs were also detected in female animals of 15 mg/kg bw/day group.
At the detailed weekly observations, the above listed, test item related signs appeared in male (pale or cold limbs, decreased activity and piloerection) and female (pale and cold limbs and piloerection) animals of 40 mg/kg bw/day group. Pale and cold limbs were also detected in female animals of 15 mg/kg bw/day group in the course of weekly clinical observation indicating the cumulative effect of the test item.
Body weight and body weight gain
The body weight development was reduced in male and female animals of 40, 15 and 5 mg/kg bw/day groups with respect to their controls during the entire study. However the changes in 5 mg/kg bw/day group were not considered to be of toxicologically relevant due to the low degree.
Food consumption
Test item related effects on the mean daily food consumption were observed in male and female animals in 40, 15 and 5 mg/kg bw/day groups. However the changes in 5 mg/kg bw/day group were not considered to be of toxicologically relevant due to the low degree.
Reproduction
Test item related changes were detected in the reproductive performance of male and female animals administered with 40 mg/kg bw/day dose of the test item. The high dose caused infertility of female animals and probably also of male animals.
The delivery data of female animals administered with 15 mg/kg bw/day was influenced by the test item as the mean number of total births, live-borns, viable offspring and corpora lutea was significantly less and the mean post-implantation loss was higher in 15 mg/kg bw/day group with respect to the control.
Organ pathology
Smaller than normal testes, epididymides, seminal vesicles and prostate were noted for some male animals in the 40 mg/kg bw/day dose group supported by the results of organ weight evaluation (testes). Histological examination revealed decreased secretion in the tubules of the seminal vesicles with coagulating gland and prostate, decreased intensity of spermatogenesis in seminiferous tubules in the testes, and lack of mature spermatozoa in the ductuli of epididymides 40 mg/kg bw/day. In the testes, the lack of mature spermatozoa and spermatids was detectable.
Involution of corpora lutea and secondary and tertiary follicles was observed in the ovaries of all female animals belonging to the high dose group and in some animal of the 15 mg/kg bw/day group. The complete lack of corpora lutea was detected in several animals in the high dose group.

 

Offspring
There was no offspring in the 40 mg/kg bw/day group. A test item effect on the offspring development was observed in the less mean offspring weight gain in 15 mg/kg bw/day group between postnatal days 0 and 4.

 

Conclusion
Under the conditions of the present study Reaction Product of 2-Hydroxybenzoic Acid, Styrene and Oxozinc caused clinical signs, reduced body weight development and food consumption (male and female), macroscopic and weight changes accompanied by histological alterations in male genital organs and accessory sex organs, and histological alterations in female genital organ in Hsd.Brl.Han: Wistar rats after repeated dose oral administration at 40 mg/kg bw/day. The high dose caused infertility of female animals and probably also of male animals.
At 15 mg/kg bw/day, test item induced clinical signs in female animals, reduced body weight and body weight gain and reduced food consumption in male and female animals. Dam’s delivery data was slightly affected by the test item.
The offspring’s body weight development was slightly depressed between postnatal days 0 and 4.
In the 5 mg/kg bw/day group, the slight changes in the body weight and food consumption were judged to be toxicologically not relevant.
Based on these observations the No Observed Adverse Effect Levels (NOAEL) were determined as follows:
NOAEL for male rats: 5 mg/kg bw/day
NOAEL for female rats: 5 mg/kg bw/day
NOAEL for reproductive performance of the male rats: 5 mg/kg bw/day
NOAEL for reproductive performance of the female rats: 5 mg/kg bw/day
NOAEL for F1 Offspring: 5 mg/kg bw/day

Justification for classification or non-classification

According to the CLP Regulation (Annex I of 1272/2008/EC), the substance Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc is classified as: Reproductive toxicity Category  1B. In a study conducted on rats, Reaction Product of 2-Hydroxybenzoic Acid, Styrene and Oxozinc caused clinical signs, reduced body weight development and food consumption (male and female), macroscopic and weight changes accompanied by histological alterations in male genital organs and accessory sex organs, and histological alterations in female genital organ in Hsd.Brl.Han: Wistar rats after repeated dose oral administration at 40 mg/kg bw/day. The high dose caused infertility of female animals and probably also of male animals, which fulfills the criteria of Category 1B.