Dialuminium zinc tetraoxide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproduction/Developmental toxicity screening test OECD 421 (oral, rat):

NOAEL (systemic) = 1000 mg/kg bw/day for both male and female parental animals

NOAEL (fertility) = 1000 mg/kg bw/day for both male and female parental animals

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 26, 2016 - August 15, 2018

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)

Version / remarks:

Adopted: July 27, 1995

Deviations:

yes

Remarks:

No analysis of dose formulations was performed as the test substance is a poor soluble inorganic substance, that is stable under the conditions. Dose formulations were freshly prepared every day.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Beijing Vital River Laboratory Animal Co., LTD.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 - 10 weeks
- Weight at study initiation: 302.7 to 345.7 g in males and 222.7 to 265.5 g in females
- Housing: Animals were housed in PP-4 type cage (L x W x H：400 mm x 250 mm x200 mm) using crop cob as bedding material.
Maximum 5 animals per cage were housed during quarantine and acclimatization period, and maximum 2 animals per cage after group assignment. During mating, 1 male and 1 female per cage were housed in the bottom free M-5 cage (L x W x H: 400 mm x 260 mm x 200 mm). Post-mating, males were housed in their home cage and females were individually housed during gestation period.

- Diet: Pelleted rodent diet (Shanghai Puluteng Bio-technology Co., Ltd, Feed Production Certificate No: Hu Si Zheng (2014) 04001), Lot. No.: M01-F-20160711026 (valid till Jan 10, 2017); M01-F-20161227047 (valid till Jun 26, 2017); ad libitum
- Water: Drinking water for labour animals; ad libitum
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY: The bedding material evaluation for contaminants was performed according to the national standard GB 14924.2-2001. The food evaluation for nutrients was performed by the supplier according to the Chinese national standard GB 14924.3-2010, for contaminants according to GB 14924.2-2001. The total bacteria numbers evaluation of water was performed according to the national standard for drinking water GB 5749-2006 monthly in the facility and the evaluation for contaminations was performed twice a year by the supplier according to GB 5749-2006. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.12 to 24.94, automatically controlled by air conditioner (Honeywell EBI)
- Humidity (%): 40.88 to 67.09
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

sterilized water for injection

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
Appropriate amounts of the test item were weighted into a tared glass and the vehicle (water for injection) was added to obtain the designed final concentration of the test item formulations. Homogeneity of the test item in the vehicle was maintained by stirring the prepared suspension thoroughly before administration. The vehicle (water) was used as the control item.
The test item formulations were freshly prepared every day prior to application.

VEHICLE
- Amount of vehicle: 10 mL /kg bw
- Lot/batch nos.: M16091803, M16100715, M16110808, M16110715

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 5 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

no

Remarks:

No analysis of dose formulations was performed as the test substance is a poor soluble inorganic substance, that is stable under the conditions. The dose formulations were freshly prepared every day and continually mixed duirng the application.

Duration of treatment / exposure:

Males were treated from 14 days prior to mating and for 56 days (8 weeks) till the day of the scheduled necropsy.

Females were treated from 14 days prior to mating up to 4 days after delivery. Females which failed to deliver were continually treated until the 25th day of pregnancy.

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

No applicable for an reproduction/developement screening test (OECD 421)

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: toxicological information: LD50 (oral) > 2000 mg/kg bw and no signs of toxicity at acute sublethal concentration: The results of a previous 28-day toxicity test, in SD rats were orally treated with zinc aluminium oxide (D-Prism) the dose levels of 100, 300, 1000 mg/kg bw once a day for 28 days. The results of the study revealed no toxicological signs in clinical observation, body weight, food intake, and there were no treatment related changes noted in the haematological, clinical chemistry, gross pathological and histopathological examinations. NOAEL of this study was greater than 1000 mg/kg bw. Therefore, based on results of the 28-day repeated dose toxicity study, in the present study dose levels of 100, 300 and 1000 mg/kg bw/day were selected.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Cage side observations included: twice daily (once in the morning, once in the afternoon) for mortality, food and water intake

The maternal behaviour including cleaning, nursing, nesting behavior and lactation was observed during the delivery period.

DETAILED CLINICAL OBSERVATIONS: once a week for viability clinical signs, behaviour, mental state, glandular secretion, respiration status, faecal traits and colon, genitals, mortality and other toxic manifestations

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed twice a week. Females were weight twice a week prior to successfully mating. During pregnancy, females were weighed on Day 0, 3, 7, 10, 14, 17, 20, and on the Day 0 of parturition and Day 4 of post-partum.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: Male food consumption was determined once a week except the mating days; Females were determined once a week prior to successfully mating. During pregnancy, female food consumption was determined on Day 0, 3, 7, 10, 14, 17, 20, and on the Day 0 of parturition and Day 3 of post-partum.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

Oestrous cyclicity (parental animals):

No

Sperm parameters (parental animals):

Parameters examined in male parental generations:
Testis weight, epididymis weight and seminal vesicles weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: Yes

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, exterior deformation, postnatal mortality and body weights (Day 0 and 4)

GROSS EXAMINATION OF DEAD PUPS: No

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE: Yes
Males: All males were sacrificed after administration on Day 56.
Females: Parturient females were sacrificed on Day 4 post-partum; non-parturient females were killed on Day 26 of pregnancy. After sacrifice, all animals were subjected to a full post mortem necropsy and histopathological examinations.

GROSS NECROPSY: Yes
- All parent animals were subjected to a full post mortem necropsy of external and internal examinations including the cervical, thoracic and abdominal viscera.
- At the time of sacrifice, all adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system as below:
Males: brain, heard, liver, spleen, lung, kidney, testes, epididymides, seminal vesicles, prostate;
Parturient females: brain, heart, liver, spleen, lung, kidney, uterus, ovaries and number of corpor lutea and implantation
Non-parturient females: brain, heart, liver, spleen, lung, kidney, uterus, ovaries, and number of dead embryos by staining of uterus with ammonium sulphite, if non-implantation was observed.

ORGAN WEIGHTS: Yes
The body weights of animals were recorded on the day of necropsy.
The weight of brain, uterus and ovaries, testes, epididymides, prostate and seminal vesicles were determined. Based on the absolute organ weight, ratios of both organ to body and organ to brain were calculated.

HISTOPATHOLOGY: Yes
Histopathological examinations were performed on ovaries and uterus, testes, epididymides, seminal vesicles and prostate of the highest dose group (1000 mg/kg bw) and control group. Examinations were not extended to the animals of other dosage groups, because no changes were seen in the highest dose group.

Postmortem examinations (offspring):

SACRIFICE
- All offspring were sacrificed at 4 days of age.


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

Statistics:

The following parameters were subject to statistical analysis:
Body weight, body weight gain, food consumption, absolute and relative organ weight, number of days required for copulation, copulation index, offspring delivered, number of corpora lutea and implantations, total number of absorbed fetuses, number of stillbirths and live offspring, body weight of offspring on postnatal days 0 and 4, sex rate, external anomalies, body surface temperature of offspring and milk stain.

Levene’s test was used to analyze the variance homogeneity of quantitative data. In the case of homogeneity of variance (P>0.05), they were evaluated using one-way analysis of variance (ANOVA). In the case of heterogeneity of variances (P≤0.05), Kruskal-Wallis (K-W) H tests were used to analyze. And if ANOVA was with significant difference (P≤0.05), Dunnett’s t test (Dunnett) was then used for pairwise comparisons; If ANOVA was not significant (P>0.05), the statistical analysis was completed. When K-W H tests were significant (P≤0.05), Mann-Whitney (M-W) U tests were used for pairwise comparisons. If K-W H tests were not significant (P>0.05), the statistical analysis was completed.

Fisher’s exact probabilities test (Fisher EXACT) was utilized to analyze qualitative data.

Reproductive indices:

Mating index (%) = (number of females mated / number of females paired) x 100
Fertility index (%) = (number of pregnant females / number of females mated) x 100
Gestation index (%) = (number of females bearing live pups / number of pregnant females) x 100
Pre-implantation loss rate (%) = ((number of corpus luteum - number of implantation) / number of corpus luteum) × 100
Post-implantation loss rate (%) = ((number of resorbed foetus + number of dead foetus) / number of implantations) × 100
Total loss rate (%) = (number of total loss / number of corpus luteum) × 100

Offspring viability indices:

Viability index (%) on postnatal Day 4 = (number of live offspring on postnatal day 4 /number of offspring) × 100
Live male rate (%) of live offspring = (number of live male offspring/ number of live offspring) × 100
Live female rate (%) of live offspring = (number of live female offspring/ number of live offspring) × 100
Incidence of offspring without external anomalies (%) = (number of live offspring without external anomalies / number of live offspring examined) × 100
Incidence of offspring without abnormal body surface temperature (%) = (number of live offspring without abnormal body surface temperature / number of live offspring examined) × 100
Incidence of offspring with milk stain in the stomach (%) = (number of live offspring with milk stain in the stomach / number of live offspring examined) × 100

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormalities were observed in spontaneous activity, general state, skin and hair of all groups of parental rats. No abortion or premature birth was observed in all pregnant rats.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No mortalities were observed in parental rats of all groups during the entire period of the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Result:
Body weight and body weight gain:
Males: Very slight decrease of body weight (3%) after mating at 1000 mg/kg bw/day in comparison to the control, but no statistic significance. Otherwise, no adverse changes in body weights and body weight gain were noted up to 1000 mg/kg bw/day.

Females:
No effects on body weights or body weight gain were noted throughout the study period up to 1000 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

On Day 9 of administration, food intake of females at 1000 mg/kg bw/day was significantly lower than that of control group (P≤0.05) and on gestation day 7, feed intake of pregnant females at 1000 mg/kg bw group was significantly higher than that of control group (P≤0.05), but the difference was small (less than 15%). These variations are considered to have no toxicological significance.
No other variations in food intake during the entire period of the study.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No histopathological changes were found in testes, epididymis, prostate gland and seminal vesicle gland, uterus and ovary in males and females of all of treatment groups when compared with control group (P>0.05).

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

No mortalities were observed in pregnant animals during the entire period of the study. All the parental animals mated successfully gave birth within 24 days of pregnancy. Mating index was considered not to be affected by treatment up to 1000 mg/kg bw/day. All females showed evidence of mating. Precoital time was not considered to be affected by treatment.
Fertility index was not considered to be affected by treatment up to 1000 mg/kg bw/day. All mated females at 1000 mg/kg bw/day were pregnant.
One mated female at 100 mg/kg bw/day and three at 300 mg/kg bw/day were not pregnant (P>0.05), while all females at 1000 mg/kg bw/ day were pregnant. Gestation index was not considered to be affected by treatment up to 1000 mg/kg bw/day.

No obvious changes were found in the number of corpora lutea, number of implantations, pre-implantation loss rate of pregnant rats in treatment groups when compared with control group, (P>0.05). No statistically significant difference was found in reproductive indices in treatment groups when compared to control group. No changes in organ weight, ratios of organ to body weight and organ to brain in males and females of all treatment groups, and no changes in pathological examinations were observed.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse and treatment-related effects were observed at 1000 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

No abnormalities were observed in spontaneous activity, general state, skin and hair of all groups of F1 generation rats.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Stillborn, missing or accidental death (all pups of one dam at 300 mg/kg bw/day were dead within Day 4 post-partum) were found in some F1 rats in the control group and treatment groups, but there was no significant difference in number of stillbirths and viability index of post-partum Day 0 and 4 among these groups (p>0.05). The details see table 5.

The total number of resorbed foetuses, number of stillbirths was marginally increased at 300 and 1000 mg/kg bw/day. Post-implantation loss and total loss rate at 300 and 1000 mg/kgbw/day were biologically although not statistically significantly increased. Also the resulting number of live offspring at 300 and 1000 mg/kgbw/day was decreased when compared with the control group (without reaching statistical significance, P>0.05) (see Table 2). It is considered that these differences are related to treatment.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No obvious abnormal changes were found in the body weight of all groups of F1 generation rats during the lactation days (P>0.05).

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no gross pathological changes found in heart, liver, spleen, lungs, kidneys and brain of F1 generation rats necropsied.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

When compared with control group, no obvious changes were found in the number of males and females, sex rate, viability index, incidence of offspring without external anomalies, incidence of offspring without abnormal body surface temperature and incidence of offspring with milk stain in the stomach (P>0.05).

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

100 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

Relevant for humans:

not specified

Table 1 Summary report of effects on copulation indices of females

Group	Copulation successful days		Number of paired females	Number of females with successful copulation	Copulation index (%)
	n	mean±SD
Control	12	3.2 ± 1.4	12	12	100
D-PrisM 100 mg/kg	12	2.8 ± 1.3	12	12	100
D-PrisM 300 mg/kg	12	2.4 ± 1.1	12	12	100
D-PrisM 1000 mg/kg	12	2.3 ± 0.9	12	12	100

Table 2 Summary report of effects on reproduction indices of females (1)

Index	Control		D-PrisM 100 mg/kg		D-PrisM 300 mg/kg		D-PrisM 1000 mg/kg
	n	mean±SD	n	mean±SD	n	mean±SD	n	mean±SD
Number of corpora lutea	12	18.3 ± 1.4	10	17.9 ± 1.4	9	17.6 ± 1.4	12	16.5 ± 4.4
Number of implantations	12	17.7 ± 1.7	10	17.3 ± 1.6	9	17.0 ± 1.7	12	16.0 ± 4.9
Total number of absorbed fetuses	12	1.1 ± 1.3	10	1.2 ± 1.1	9	2.0 ± 2.1	12	3.6 ± 3.9
Number of stillbirths	12	0.2 ± 0.4	10	0.0 ± 0.0	9	0.6 ± 1.1	12	0.7 ± 2.3
Number of live offspring	12	16.4 ± 1.5	10	16.1 ± 1.6	9	14.4 ± 3.5	12	11.8 ± 6.4
Pre-implantation loss rate (%)	12	3.3 ± 2.9	10	3.4 ± 4.2	9	3.2 ± 3.1	12	4.4 ± 6.9
Post-implantation loss rate (%)	12	6.8 ± 7.6	10	6.8 ± 5.9	9	15.7 ± 15.9	12	26.7 ± 30.3
total loss	12	1.8 ± 1.4	10	1.8 ± 1.5	9	3.1 ± 2.4	12	4.8 ± 5.2
total loss rate (%)	12	9.9 ± 7.1	10	9.9 ± 7.7	9	18.5 ± 15.6	12	29.9 ± 30.5
Parturition days	12	23.2 ± 0.6	10	23.1 ± 0.3	9	23.4 ± 0.5	12	23.1 ± 0.3

Table 3 Summary report of effects on reproduction indices of females (2)

Group	Number of females with successful copulation	Number of pregnant rats	Number of females with absorbed fetuses	Number of females with stillbirths	Number of females with live offspring	Percentage of females with absorbed fetuses (%)	Percentage of females with stillbirths (%)	Gestation index (%)	Fertility index (%)
Control	12	12	7	2	12	58.3	16.7	100.0	100.0
D-PrisM 100 mg/kg	12	10	8	0	10	80.0	0.0	100.0	83.3
D-PrisM 300 mg/kg	12	9	8	2	9	88.9	22.2	100.0	75.0
D-PrisM 1000 mg/kg	12	12	10	1	11	83.3	8.3	91.7	100.0

Table 4 Summary report of effects on development indices of F1 generation rats

Index	Control		D-PrisM 100 mg/kg		D-PrisM 300 mg/kg		D-PrisM 1000 mg/kg
	n	mean±SD	n	mean±SD	n	mean±SD	n	mean±SD
Number of females	12	8.7 ± 2.6	10	8.9 ± 1.7	9	8.2 ± 2.0	11	7.0 ± 3.5
Number of males	12	7.8 ± 2.1	10	7.2 ± 2.3	9	6.2 ± 3.5	11	5.8 ± 3.1
Sex rate (females, %)	12	52.4 ± 13.8	10	55.8 ± 11.9	9	59.9 ± 18.1	11	54.8 ± 20.0
Sex rate (males, %)	12	47.6 ± 13.8	10	44.2 ± 11.9	9	40.1 ± 18.1	11	45.2 ± 20.0
Viability index of lactation day 0 (%)	12	100.0 ± 0.0	10	100.0 ± 0.0	9	100.0 ± 0.0	11	100.0 ± 0.0
Viability index of lactation day 4 (%)	12	96.4 ± 9.0	10	98.9 ± 3.5	9	82.9 ± 32.6	11	100.0 ± 0.0
Incidence of offspring without external anomalies(%)	12	100.0 ± 0.0	10	100.0 ± 0.0	9	100.0 ± 0.0	11	100.0 ± 0.0
Incidence of offspring with milk stain (%)	12	100.0 ± 0.0	10	100.0 ± 0.0	9	100.0 ± 0.0	11	100.0 ± 0.0
Incidence of offspring without abnormal body surface temperature (%)	12	100.0 ± 0.0	10	100.0 ± 0.0	9	100.0 ± 0.0	11	100.0 ± 0.0

Table 5 Summary report of the death data of F1 generation rats

Group		missing		accidental death		stillborn		Total death
Control		6		1		2		9
D-PrisM 100 mg/kg		2		0		0		2
D-PrisM 300 mg/kg		18*		1		5		24
D-PrisM 1000 mg/kg		0		0		8		8

*All the 14 F1 rats in the litter of 3F012 females were missing, which might be eaten by the dam. The data of this nest were not included in the statistical analysis.

Conclusions:

The NOAEL of test substance was considered to be 1000 mg/kg bw/day for general systemic toxicity of both male and female parental animals;
The NOAEL of test substance was considered to be 1000 mg/kg bw/day for reproductive performance of both male and female parental animals;
The NOAEL of test substance was considered to be 100 mg/kg bw/day for development of offspring.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.7, of Regulation (EC) No. 1907/2006.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A reproduction/developmental toxicity screening test was conducted according to OECD 421 and in compliance with GLP (Wang, 2018). Based on the results of a previous 28-day repeated dose toxicity study, groups of each 12 males and 12 females of SD rats were treated once daily by gavage with the test substance at doses of 0 (control: injection water), 100, 300, and 1000 mg/kg bw/day for investigation of the effects of its repeated administration to males and females, the effects on the reproduction of males and females, and on development of offspring. Males were treated for 2 weeks prior to mating, during mating, and up to termination for 56 days (8 weeks). The females were treated from 2 weeks before copulation to post-partum Day 4. The females that delivered were treated for 2 weeks prior to mating, during mating, during post-coitum, and to post-partum Day 4. Females that failed to deliver healthy offspring were treated till Day 26 of pregnancy.

 

No mortality or adverse effects were noted in the systemic toxicity in all of the parental animals up to the dose of 1000 mg/kg. No abortion or premature birth was observed in all pregnant females. Male rats, parental and pregnant rats, as well as live F1 pups of all treatment groups were generally in good condition, with normal autonomic activities, good mental state, clean fur and no bristles or other toxic symptoms caused by treatments. Slight fluctuations of body weight of males were noted in the late treatment stage (3%) while body weight development of females was not affected by treatment up to 1000 mg/kg bw/day. Significant changes in food intake were observed in females on the day 9 of administration, and on gestation day 7 when compared to the control (P≤0.05), but the difference was small (less than 15%). These abnormalities are considered to have no toxicological significance. No other abnormalities in food intake during the entire period of the study. There were no significant differences observed in absolute and relative organ weight. No changes were found in gross pathological and histopathological examinations on the organs including the organs of reproduction systems of males and females.

 

There were no toxicological relevant changes in the mating index, fertility and gestation index.

In conclusion, as no adverse effects were observed for systemic or fertility, the no-observed-adverse effect level was considered to be 1000 mg/kg bw/day for systemic toxiciy and fertility.

Effects on developmental toxicity

Description of key information

Reproduction/Developmental toxicity screening test OECD 421 (oral, rat):

NOAEL (parental toxicity) = 1000 mg/kg bw/day

NOAEL (developmental toxicity) = 100 mg/kg bw/day

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.7, of Regulation (EC) No. 1907/2006.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

A reproduction/developmental toxicity screening test was conducted according to OECD 421 and in compliance with GLP (Wang, 2018). As described above, no adverse effects were observed regarding systemic toxicity and fertility of the parental animals.

F1 pups of all treatment groups were generally in good condition, with normal autonomic activities, good mental state, clean fur and no bristles or other toxic symptoms caused by treatments.

No changes from controls were observed in body weight of F1 pups. There were no significant differences observed in F1 pups (viability index on postnatal Day 4, live male rate, live female rate, incidence of offspring, incidence of offspring without abnormal body surface temperature, incidence of offspring with milk stain in the stomach) when compared to control, and no changes in macroscopic examination of pups killed on Day 4 after birth.

The total number of resorbed foetuses, number of stillbirths, post-implantation loss rate and total loss rate at 300 and 1000 mg/kg bw/day groups were increased, and the number of live offspring was decreased when compared with the control group, although they all were not statistically significant (P>0.05). However, the mean value of number of stillbirths, post-implantation loss rate, total loss and total loss rate at 300 and 1000 mg/kg bw/day were twice or more higher than that of control group, and the stillborn of 300 and 1000 mg/kg bw/day groups were obvious higher than the control group, so it is considered that these differences are related to the test substance. Therefore, the NOAEL for developmental toxicity is considered to be 100 mg/kg bw/day.

Mode of Action Analysis / Human Relevance Framework

Not applicable

Justification for classification or non-classification

The available reproduction toxicity screening study reveals that the treatment with zinc aluminium oxide induced adverse effect in prenatal stage due to an increased incidence of post-implantation loss and subsequently reduced litter size at 300 and 1000 mg/kg bw/day. A classification for reproductive toxicity (H361) at this stage is not warranted because the effects were not considerd severe, i.e. no statistical significance and pathological examinations did not reveal any changes when compared to control. Also, all females in the high dose group had implantation sites.

Based on the available data on reproductive toxicity, there is indication that zinc aluminium oxide induces developmental toxcity. However, no final decision on classification for toxicity to reproduction according to Regulation (EC) No. 1272/2008 can be made, as only a screening study according to OECD 421 is available.