Zinc acrylate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Reference 1

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Well-documented study report, comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Principles of method if other than guideline:

10 male and 20 female rats per group were administered acrylic acid at dosage goals of 0, 83, 250 and 750 mg/kg/bw/day in drinking water. At the end of 13 weeks, the male and female rats from each group were mated one male to two females for a 15-day period. Females were introduced into male cages. Rats ware assigned to treatment groups using a computer generated random number scheme. The F0 generation rats were sacrificed after weaning of the F1 generation and were approximately 194 days old at the time of sacrifice. The F1 generation rats were sacrificed at 21 days of age. Treatment effects were determined by statistical comparison of mortality, body weight change, food and water consumption, organ weight change and histological evaluation of tissues from sacrificed animals.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Microbiological Associates, Inc., Walkersville, MD.
- Age at study initiation: (P): 41 days
- Weight at study initiation: (P) Males: 111-139 g; Females: 85-118 g
- Housing: During mating, two females were placed in a cage with one male; at this time all rats received acrylic acid at the concentration in the water for the respective female groups. Fifteen days after the first mating, cohabitation was discontinued and the individual females were placed in plastic cages fitted with wire rod metal tops. Ab-sorb-dri hardwood chips were used for bedding in the plastic cages.
- Diet (ad libitum): Zeigler Brothers NIH-07
- Water (ad libitum): Tap water


ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Photoperiod (hrs dark / hrs light): no data

Route of administration:

oral: drinking water

Vehicle:

water

Details on mating procedure:

- M/F ratio per cage: 1/2
- Length of cohabitation: 15 days
- After successful mating each pregnant female was caged (how): Fifteen days after the first mating, cohabitation was discontinued and the individual females were placed in plastic cages fitted with wire rod metal tops.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of aqueous solutions of acrylic acid was determined using a gas chromatographic procedure. The mean dosage levels attained were quite close to the dosage goals for each group. The mean overall dosages of acrylic acid were 0.73, 0.25 and 0.085 g/kg/bw/day for males and 0.72,
0.25 and 0.083 g/kg/bw/day for females.

Duration of treatment / exposure:

Exposure period: 13 weeks
before the mating and afterwards during the gestation and lactation periods.
Premating exposure period (males): 13 weeks
Premating exposure period (females): 13 weeks
Duration of test: approx. 6 months

Frequency of treatment:

continuously

Remarks:

Doses / Concentrations:
0, 83, 250, 750 mg/kg bw/d
Basis:
nominal in water

No. of animals per sex per dose:

10 males and 20 females

Control animals:

yes, concurrent vehicle

Positive control:

none

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS:
Daily observation for clinical signs

BODY WEIGHT:
Individual body weights for each F0 animal were determined weekly.

FOOD CONSUMPTION:
The food consumption rate was recorded weekly. Fresh diet was added to the jars every week.

WATER CONSUMPTION:
The water consumption rate was recorded weekly. Fresh solutions were prepared each week, with the percentage of acrylic acid in the water (X) adjusted to maintain a relatively constant dosage level (K) in g/kg according to formula:

X = 100 KW / G

Where X and K are explained above:
W = predicted mean body weight, kg
G = mean water consumption, ml

Oestrous cyclicity (parental animals):

not examinated

Sperm parameters (parental animals):

not examinated

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
Five male and five female rats, randomly selected from each dosage level of the F0 parents, were anesthetized with methoxyflurane and sacrificed by severing the brachial vesseis to permit exsanguination.


GROSS NECROPSY
- All sacrificed rats were given a complete gross necropsy examination and organ weights were recorded for the liver, kidneys, heart, spleen, brain and testes.


HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were taken and fixed in 10% neutral buffered formalin, processed for paraf in embedding, sectioned at 5 microns and stained with hematoxylin and eosin on all high dose and control animals. Only those tissues with gross lesions were examined microscopically from the intermediate and low level animals.
Pituitary, thyroids, parathyroids, adrenals, heart, thymus, spleen, testes, epididymides, kidneys, urinary bladder, tongue, submandibular salivary gland, esophagus, stomach, duodenum, colon, mesenteric lymph node, nasal cavity, trachea, lungs, ovaries, oviduct, liver, pancreas, brain, eyes, skin, mammary gland, sternum, any lesions.

Postmortem examinations (offspring):

SACRIFICE
Five male and five female rats, randomly selected from each dosage level of the F1 weanlings, were anesthetized with methoxyflurane and sacrificed by severing the brachial vesseis to permit exsanguination.


GROSS NECROPSY
- All sacrificed rats were given a complete gross necropsy examination and organ weights were recorded for the liver, kidneys, heart, spleen, brain and testes.


HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were taken and fixed in 10% neutral buffered formalin, processed for paraf in embedding, sectioned at 5 microns and stained with hematoxylin and eosin on all high dose and control animals. Only those tissues with gross lesions were examined microscopically from the intermediate and low level animals.
Pituitary, thyroids, parathyroids, adrenals, heart, thymus, spleen, testes, epididymides, kidneys, urinary bladder, tongue, submandibular salivary gland, esophagus, stomach, duodenum, colon, mesenteric lymph node, nasal cavity, trachea, lungs, ovaries, oviduct, liver, pancreas, brain, eyes, skin, mammary gland, sternum, any lesions.

Statistics:

For every experimental parameter or index measured, the results of each of the three test levels were compared with the control group. To evaluate the statistical significance of possible changes in continuous data, the analysis of variance (ANOV) validated by Bartlett's test for homogeneity of variance, was used. Individual mean differences were identified by Duncan's multiple range test when indicated by a significant F value for ANOV. In the case of heterogeneous variances, as indicated by Bartlett's test, the paired group F test, and either the Cochran or the Student t-test were used to identify significant differences. Enumerative data were evaluated statistically by NXR Chi Square test; differences between groups were delineated by Fisher's Exact test. Non-parametric data were compared by a distribution-free multiple comparison method. The fiducial limit of 0.05 was employed as the critical level of difference not attributable to chance

Reproductive indices:

The following reproductive parameters were evaluated statistically:
1. Fertility index - the proportion of females that were pregnant of the number that were mated or the proportion of the males shown to be fertile of the number that were mated.
2. Gestation Index - the proportion of pregnancies that resulted in litters with live pups.
3. Gestation Survival Index - the proportion of newborn pups that were alive at birth.
4. Pups born alive per litter.
5. Days from mating to litter.

Offspring viability indices:

The following reproductive parameters were evaluated statistically:
1. 5-Day Survival Index - the proportion of liveborn that survived 5 days.
2. 21-Day Survival Index - the proportion of pups retained on day 5 that survived 21 days.
3. Pups weaned per pups alive at birth.

Clinical signs:

no effects observed

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

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
There were no significant abnormal clinical signs observed. No deaths occurred during the study.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS):
Body weight gain was depressed markedly for both sexes at the high dosage level. This effect was statistically highly significant in these groups throughout the study. At the highest dosage level there were effects on diet and water consumption, body weight gain and organ weights (liver, kidneys, spieen, testes, brain). At the intermediate dosage level, a decrease in water consumption was noted for both sexes. Body weight gain was reduced while liver and kidney weights (absolute and/or relative) were increased for the female rats. At the lowest dosage level, absolute liver and kidney weights and relative liver weights were increased for the females. These changes were possibly chemically induced.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS):
At the highest dose, a numerical reduction of the fertility (male and female) and gestation indices, pups born alive and pups weaned was observed.


ORGAN WEIGHTS (PARENTAL ANIMALS):
The effect of acrylic acid on the organ weights was obvious at the high dosage level in both sexes. In the male rats this effect included: Significant reduction in absolute liver weight and significant increase in relative kidney, spleen and testes weights.
In the female rats, the effects on organ weights at the high dose level included: Significant reduction in absolute liver and spleen weights and significant increase in relative kidney and brain weights. Furthermore, the significant increases in relative liver weight and absolute and relative kidney weights for the intermediate level females are considered to be dose-related effects. The significant increase in absolute liver and kidney weights and relative liver weights in the lowest dosage level females are possibly chemically-induced changes. However, the significant increase in female fertility in this group precludes the probability that reproductive behavior was affected by these changes. They are, therefore, considered to be of no biological importance on reproduction.


GROSS PATHOLOGY (PARENTAL ANIMALS):
There were no gross lesions which could be attributed to treatment with acrylic acid.

HISTOPATHOLOGY (PARENTAL ANIMALS):
Histopathological examination revealed no treatment-related lesions associated with the ingestion of acrylic acid.

Key result

Dose descriptor:

NOAEL

Effect level:

83 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: Body and organ weights.

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: fertility index; gestation index

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

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:

no effects observed

CLINICAL SIGNS (OFFSPRING):
There were no significant abnormal clinical signs observed. There were no consistent findings in the spontaneous death of neonatal rats.


BODY AND ORGAN WEIGHT (OFFSPRING)
The effects at the highest dosage level of the parent generation were similar to those observed for the highest dosage level of the progeny. Here
also, there were effects on body weight gain and organ weights (liver, heart, kidneys, brain, spleen).


GROSS PATHOLOGY (OFFSPRING):
There were no gross lesions which could be attributed to treatment with acrylic acid.


HISTOPATHOLOGY (OFFSPRING)
Histopathological examination revealed no treatment-related lesions associated with the ingestion of acrylic acid.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

250 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: Pup body and organ weights; number of pups born alive and pups weaned

Key result

Reproductive effects observed:

not specified

Mean body weight changes (g)

Week of treatment	0 mg/kg/bw/d		83 mg/kg/bw/d		250 mg/kg/bw/d		750 mg/kg/bw/d
	male	female	male	female	male	female	male	female
Mean bw at day 0	124.5	99.4	124.0	101.3	125.3	102.4	121.4	99.0
1	29.8	12.7^x	28.6	14.2	31.2	13.8^x	24.1^a	10.6^a
2	62.2	26.8	56.8	27.6	63.1	23.5^a	51.3^b	21.4^c
3	89.2	37.6	83.4	38.1	91.7	36.6	75.7^b	30.0^c
4	112.2	47.6	106.5	46.7	111.1	45.4	93.6^b	36.9^c
5	130.3	55.6	126.7	55.8	128.6	52.5	106.7^b	41.6^c
6	145.3	63.6	142.2	63.8	141.8	58.6^a	119.0^c	47.2^c
7	159.5	69.4	154.2	70.3	153.0	63.6^a	127.8^c	49.8^c
8	171.8	73.3	167.7	74.1	162.6	67.1^a	125.6^c	52.1^c
9	181.1	75.9	176.4	77.2	170.1	69.2^a	131.6^c	53.0^c
10	191.4	79.9	187.6	81.0	180.0	73.3^a	139.2^c	55.2^c
11	200.1	82.4	194.9	83.3	185.4	74.5^b	142.8^c	57.4^c
12	205.2	84.4	200.6	86.0	191.7	78.0^a	144.7^c	58.6^c

^a0.05>p>0.01

^b0.01>P>0.001

^cP<0.001

^xOne clinically ill rat excluded from statistical evaluation of body weight gain.

Reproductive Performance

Parameter	Dose (mg/kg/bw/d
	0	83	250	750
Fertility index (males)^a	80	100	80	60
Fertility index (female)^b	50	95	75	45
Gestation index^c	100	100	100	89
Gestation survival index^d	100	100	100	100
5-Day survival index^e	100	100	100	100
21-Day survival index^f	100	100	100	100
Pups born alive/Litter^g	6	8	9	4
Pups weaned/Pups alive at birth (100)^g	100	100	100	42

 ^aLitters sired per males mated x 100

^bDeliveries per females mated x 100

^cLitters with live pups/total pregnancies x 100

^dPups born viable/total pups delivered x 100, median per litter

^ePups viable at day 5/pups born viable x 100, median per litter

^fPups viable on day 21/pupe retained at day 5 x 100, median per litter

^gMedians

Conclusions:

At the highest dosage level, the preponderance of statistical significance in many parameters in both parents and progeny concomitant with generally lowered reproductive indices is a clearly dose-related toxic effect. Therefore, based on the above findings the maximum dosage level that did not produce a deleterious reproductive effect for one generation of exposure of acrylic acid in the drinking water of rats was estimated to be 250 mg/kg bw/day (equivalent to 720 mg zinc diacrylate/kg/day).

Executive summary:

A study was conducted to evaluate the reproductive toxicity potential of the test substance in rats for one generation. The method was equivalent to OECD Guideline415. 10 male and 20 female rats per group were administered acrylic acid at dose goals of 0, 83, 250 and 750 mg/kg/bw/day in drinking water. At the end of 13 weeks, the male and female rats from each group were mated one male to two females for a 15 d period. The F0 generation rats were sacrificed after weaning of the F1 generation and were approximately 194 d old at the time of sacrifice. The F1 generation rats were sacrificed at 21 d of age. Treatment effects were determined by statistical comparison of mortality, body weight change, food and water consumption, organ weight change and histological evaluation of tissues from sacrificed animals. Deleterious effects were observed at the high dose level in both sexes for parents and progeny. These effects included diet and water consumption, body and organ weight changes. Furthermore, there was a numerical reduction in the fertility and gestation indices and the number of pups weaned. At the intermediate level, although the reproductive parameters were equivalent to those of the controls, dose-related effects were seen for water consumption and body and organ weights in the parent rats. At the low dose level, organ weight changes observed in the female rats of the parent generation are considered to be of minimal biological significance. The results of the reproduction study do not indicate a substantial deleterious effect of acrylic acid on reproductive performance, since there were no statistically significant differences among the treated and control groups. However, the data should be interpreted cautiously because of a relatively atypical control group. For example, high dose females had relatively low fertility (45%) but this value was equivalent to that of the controls (50%). Although the average fertility index for earlier Fischer rat studies was 82%, the occurrence of a concurrent control group with lower fertility makes it impossible to determine whether the low fertility of the high dose females was treatment-related. Similarly, the typical (median) historical control value of 9 pups per litter for Fischer rats differs substantially from the control median of 6 in this study. Therefore, reduced litter size at the high dose level may or may not have been a treatment-related effect. In spite of the difficulty in interpreting the possible reproductive effects of acrylic acid at the high dose level, the results of the reproduction study indicate that there was no adverse effect at the two lower levels. This conclusion is justified whether one uses the concurrent control data or historical control data as basis for comparison. In summary, although numerous treatment-related effects were observed at the two highest dose levels, many of these may be the result of reduced food and water consumption rather than specific toxic effects of acrylic acid. At the highest dose level, the preponderance of statistical significance in many parameters in both parents and progeny concomitant with generally lowered reproductive indices is a clearly dose-related toxic effect. Therefore, based on the above findings the maximum dose level that did not produce a deleterious reproductive effect for one generation of exposure of acrylic acid in the drinking water of rats was estimated to be 250 mg/kg bw/day (equivalent to 720 mg zinc diacrylate/kg/day) (IATG, 1980).

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From May 19, 1992 to April 19, 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Wistar rats (Chbb = THOM (SPF))
- Source: Karl THOMAE, Biberach an der Riss, Germany
- Age at study initiation: (P): 35 ± 1 days
- Weight at study initiation: (P) Males: 140.0 (127 - 154) g; Females: 118.8 (106 - 130) g
- Housing: Single in type DK III stainless steel wire mesh cages, with the following exceptions: during mating periods, the males designated for mating were kept individually in Makrolon cages, type M III; for the overnight mating the females were put into the cages of the males. From day 18 of pregnancy until day 14 after birth, the pregnant animals and their litters were also housed in Makrolon type M III cages.
- Diet (ad libitum): Kliba maintenance diet rat/ mouse/hamster GLP 343 meal (KLINGENTALMUEHLE AG, Kaiseraugst, Switzerland)
- Water (ad libitum): Tap water
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: drinking water

Vehicle:

water

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: a maximum of 3 weeks.
- Proof of pregnancy: sperm in vaginal smear referred as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The content of Acrylic acid in the aqueous solutions was determined by gas chromatography.

Duration of treatment / exposure:

Exposure period: F0: at least 70 days before the mating and afterwards during the gestation and lactation periods.
F1: at least 98 days before the mating and afterwards during the gestation and lactation periods.
Premating exposure period (males): at least 70 days
Premating exposure period (females): at least 70 days
Duration of test: approx. 12 months

Frequency of treatment:

continuously

Remarks:

Doses / Concentrations:
0, 500, 2500 and 5000 ppm (corresponding to approx. 0, 53, 240 and 460 mg/kg bw/day)
Basis:
nominal in water

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Positive control:

none

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS:
All parental animals were checked daily for clinically evident signs of toxicity. Particular attention was given to the nesting, littering and lactation behaviour of the dams, but only special findings were documented.

BODY WEIGHT:
- Parental animals: generally, body weight was determined once weekly until the end of the study, and at the time of necropsy.
- F0 and F1 fertilized females and females with litter: body weight was determined on the day of sperm evidence in the vaginal smear and thereafter on days 7, 14 and 20 of gestation, one day after parturition, and on days 7, 14 and 21 post-parturition.
- Females without positive evidence of sperms: body weight was not determined during the mating interval.
- Females without litter: body weight was not determined during the lactation phase.

FOOD CONSUMPTION:
- F0 and F1 parental animals: food consumption was determined once weekly (over 7 days) during the period prior mating.
- Pregnant females: food consumption was determined for days 0-7, 7-14, 14-20 post coitum (pc).
- Lactating females: food consumption was determined for days 0-4, 4-7, 7-14 post parturition (pp).
- F0 and F1 dams between day 14 and 21 pp: food consumption was not determined for the F0 and F1 dams between day 14 and 21 pp, since during this period the pups started consumption of solid food; therefore there was no point in such a measurement.
- Females during mating period, females without positive evidence of sperms, females without litter: food consumption was not determined respectively during mating period, gestation period or lactation phase.


WATER CONSUMPTION:
- F0 and F1 parental animals: water consumption was determined once weekly (over 3 days) during the period prior mating.
- Pregnant females: water consumption was determined for days 0-1, 6-7, 13-14, 19-20 post coitum (pc).
- Lactating females: water consumption was determined for days 1-2, 3-4, 6-7, 13-14 post parturition (pp).
- F0 and F1 dams from day 20 and 21 pp: water consumption was not determined for the F0 and F1 dams for days 20 - 21, since during this period the pups started consumption of water; therefore there was no point in such a measurement.
- Females during mating period, females without positive evidence of sperms, females without litter: water consumption was not determined respectively during mating period, gestation period or lactation phase.


INTAKE OF TEST SUBSTANCE:
The intake of test substance (IT, in mg/kg bw/day) was calculated according to the following formula:

ITx = WCx * D / BWy

D = dose in ppm
WCx = daily water consumption on day x; in g
BWy = body weight on day y; in g

Oestrous cyclicity (parental animals):

not examined

Sperm parameters (parental animals):

not examined

Litter observations:

The pups (F1 and F2 litters) were examined as soon as on their day of birth for the determination of the total number of pups and the number of liveborn and stillborn pups (pups died on day of birth prior the first examination). Thereafter the pups were checked twice daily on workdays (once a day on week ends and public holidays) for mortality (i.e. dead and moribund pups) and the mortality (number and percentage) was determined for the day of birth (i.e. day 0) and for the periods: days 1 - 4, 5 - 7, 8 - 14 and 15 - 21 of lactation. Pups that died accidentally and had to be sacrificed because of maternal death were not considered for calculation. The number of surviving pups was determined for days 0, 4, 7, 14 and 21 of lactation and served for the calculation of the viability index and the lactation index.

The sex of the pups was determined on day 0 and day 21 (measurement of the anogenital distance, which is known to be greater in male pups than in females), and the sex ratio was calculated according to following formula:

- Sex ratio = number of live male or female pups on day 0/21 * 100 / number of live male and female pups on day 0/21

The pups were weighed on days 1, 4, 7, 14 and 21 after birth, and they were examined daily for clinical symptoms or gross morphological abnormalities. The determination of the relative organ weight was based on the pup body weight on day 21 after birth. The bodies of the sacrificed pups were examined for external abnormalities and the organs also were subjected to gross pathology; skeletal staining according to the modified Dawson´s method and/or further processing of the head according to Wilson´s method was done in case of abnormal findings. Stillborn pups as well as pups that died during weaning also were subjected to necropsy.

Development stages / Behavioral tests:
Physical development was assessed by monitoring pinna unfolding, opening of the auditory canal and opening of the eyes. Additional tests
were performed to assess grip reflex, hearing and pupillary reflex as follows :
- Grip reflex: Tested on day 13 after birth by placing front paws onto 3-mm diameter rod. For a positive response, the animal had to grip the bar and pull itself up.
- Hearing test: On day 21 after birth, animals were placed in a soundproof box and exposed to a sound (0 .1 sec, 5000 Hz, about 90 dB); a startle reflex was considered a response to this stimulus.
- Pupillary reflex: On day 2l after birth, pupillary constriction reflex was assessed by shining a penlight on the eye and observing the reaction.

Postmortem examinations (parental animals):

SACRIFICE
Parental animals were killed by decapitation under CO2 anaesthesia and examined macroscopically.

GROSS NECROPSY
Terminal body weights as well as the weights of liver, kidneys, epididymides and testes were recorded .

HISTOPATHOLOGY / ORGAN WEIGHTS
Liver, kidneys and stomach (non-glandular and glandular).

Postmortem examinations (offspring):

SACRIFICE
Pups were killed by CO2 asphyxiation, examined externally, eviscerated and their organs assessed macroscopically.


GROSS NECROPSY
External and internal examinations including the cervical, thoracic, and abdominal viscera.


HISTOPATHOLOGY
Vagina, cervix, uterus, ovaries, oviducts, testes, epididymides, seminal vesicles, coagulation gland, oesophagus and duodenum.

Statistics:

The statistical assessment of the different data obtained within the present study was based on following methods, depending on the parameters considered: Dunnett test, Fisher`s exact test and Wilcoxon test.

Reproductive indices:

Mating and fertility indices were calculated according to following formulas:

- Male mating index (%) = number of males with confirmed mating * 100 / number of males placed with females

- Male fertility index (%) = number of males proving their fertility * 100 / number of males placed with females

Remark:
Males were defined as “with confirmed mating” by the presence of vaginal sperm in the female, or by the production of a litter, or by the presence of fetuses in the uterus.
Males were defined as “proving their fertility” by female giving birth to a litter or having pups or fetuses in the uterus.

Reevaluation of fertility:
If an animal of the F0 or F1 generation parental animals had not produced any offspring after the scheduled mating of F0 parents (to get F1 litter) or after the scheduled mating of F1 parents (to get of F2 litter), those animals treated with the test substance were mated with fertile animals of the control groups. Animals of the control groups which seemed to be infertile were mated with mating partners with proven fertility of the controls.
After fertility had been reevaluated, the animals were sacrificed and subjected to gross-pathological and histopathological assessments. The uteri of the females reevaluated for fertility were examined for live and dead implantations. In the case of an apparently non-pregnant animal or of an empty uterus horn in the case of single-horn pregnancy, the uterus was stained with sodium sulfide and assessed for early implantations. Then the uteri were rinsed carefully under running water. After these examinations were completed, the uteri were transferred to the pathology lab for further fixation and evaluation.

Offspring viability indices:

- Viability index (%) = number of live pups on day 4 after birth * 100 / number of liveborn pups on the day of birth

- Lactation index (%) = number live pups on day 21 after birth * 100 / number of live pups on day 4 after birth

Remark:
Day 4 after birth preceded standardization of the litters.
Day 21 after birth followed standardization of the litters.

Clinical signs:

no effects observed

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:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

**** F0 GENERATION PARENTAL ANIMALS ****

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- There were no mortalities in any of the F0 generation parental animals in any of the groups.
- No clinical signs which might be attributed to the test substance were detected in male or female F0 generation parental animals. The 3 concentrations administered in the drinking water did not lead to disturbances of the general behavior in any of the F0 parental animals.
 - There were no particular substance-related clinical findings in F0 females during the gestation period for F1 litter. Insufficient nesting activity was observed for several dams of all groups including the controls.
 - No substance-related clinical findings were recorded for the F0 dams during the F1 lactation period.

FOOD CONSUMPTION (PARENTAL ANIMALS)
In general, the food consumption of the males (during the premating period) and of the females (during premating, gestation and lactation periods) of all test groups was not influenced by the test substance administration. It was, however, slightly, but statistically significantly reduced in the high dose males during the first week of the premating period and in the females of 5000 ppm test group during the second week of the lactation period.
The sporadic and only marginal reductions in food consumption of the 5000 ppm rats are probably related to the reduced consumption of aqueous acrylic acid solutions of these animals and thus are likely indirectly associated with the administration of the test substance. All other observable differences between the groups are without biological relevance, because they are not dose-related; this includes the statistically significantly increased food consumption of the low dose females during study weeks 0-1 and 6-7 of the premating period.

BODY WEIGHT (PARENTAL ANIMALS)
In the F0 males statistically significant reductions in mean body weights were seen in the highest dose group (5000 ppm) from week 12 until week 20 of the study period. Body weight changes of the high dose males were statistically significantly diminished only at certain study intervals (weeks 0-1, 6-7, 11-12); if calculated for the total study period (weeks 0-20), body weight gain of the high dose males was about 9% lower than that of the respective controls.
Body weights and weight gains of the substance-treated females were similar to control values during the premating period and during gestation and lactation. Only during the first week of gestation did the high dose dams gain statistically significantly less weight than the corresponding controls.
Finally the impairments in body weight/body weight gain in the high dose F0 males and - to a lesser extent - in the F0 females are assessed as being substance-related effects. All other statistically significant differences in body weights and body weight gains are considered unrelated to the test substance because the values were not influenced in a dose-dependent manner and/or are within the biological range of variation.

WATER CONSUMPTION (PARENTAL ANIMALS)
The water consumption of the high dose male and female animals was clearly reduced. This reduction was statistically significant during the premating period. It was also diminished in the 5000 ppm females during gestation and lactation of F1 litter. In total the 5000 ppm males consumed about 11% and the high dose females about 13% less drinking water (aqueous acrylic acid solutions) then the respective controls during the first 10 study weeks. The marked reduction in the drinking water consumption of the high dose rats was associated with the test substance administration. All other observable differences between the groups in respect to water consumption are without biological relevance, because they are not dose-related; this includes the statistically significantly increased water consumption of the 500 ppm female animals during premating weeks 6-7 and 9-10.

MALE REPRODUCTIVE FUNCTION (PARENTAL ANIMALS)
For all F0 males which were placed with females to generate F1 pups mating was confirmed; thus, the male mating index was 100% in all groups. For nearly all F0 males fertility could be confirmed within the scheduled mating interval; the fertility index varied between 92% and 96% with no treatment related effect. Thus, the fertility of the F0 generation parental males was not adversely influenced by the administration of aqueous acrylic acid solutions.

FEMALE REPRODUCTIVE FUNCTION (PARENTAL ANIMALS)
The female mating index calculated after the mating period for F1 litter was 100% for all groups. The mean duration until sperm was detected (day 0 pc) varied between 1.8 and 3.8 days and was statistically significantly longer for the high dose dams; the high dose value (3.8 days), however, is substantially similar to the mean cohabitation time value of the control group (3.2 days) of the second parental generation (F1 animals) and therefore was not considered treatment-related. Only one or two females in all groups, including the controls, did not become pregnant within the scheduled mating interval. The fertility index varied between 92% and 96% without any dose-response relationship. All females in question except the 2 low dose females proved to be fertile after being mated again with control males. The mean duration of gestation was similar in all groups and the gestation index reached 100% for all groups. The mean number of pups delivered/dam was uninfluenced by the test substance administered. The number of liveborn and stillborn pups was comparable between the groups, and the live birth index was 98% in test groups. Thus, the administration of aqueous acrylic acid solutions did not adversely affect reproduction and delivery data of the FO generation parental females.


GROSS AND HISTOPATHOLOGICAL FINDINGS
- Thickening of the limiting ridge (margo plicatus) of the forestomach in most male and female rats.
- Minimal hyperkeratosis at the limiting ridge of the forestomach in most male and all female rats.
- Edema in the submucosa of the glandular stomach of 2 male and 10 female rats, minimal in all cases.

**** F1 GENERATION PARENTAL ANIMALS *****

MORTALITY / CLINICAL OBSERVATIONS
- One male animal of 2500 ppm test group had to be sacrificed in a moribund state due to a severe skin lesion on the base of the tail. There were no other unscheduled mortalities in any of the test groups.
 
- No clinical signs which might have been attributed to the test substance administered were detected in male or female F1 generation parental animals. The 3 doses administered in drinking water did not lead to disturbances of the general behavior in any of the F1 parental animals. One male animal of 2500 ppm test group developed a severe skin lesion on the base of the tail and was sacrificed in a moribund state. Another male of the same dose group showed unilateral chromodacryorrhea. The clinical findings which occurred in just two intermediate dose males were spontaneous in nature.
 
- No particular clinical findings were noted for F1 dams with positive sperm detection except insufficient or no nesting activity, which was recorded for several dams of all groups (0, 500, 2500 and 5000 ppm) and which occurred without a clear dose-response relationship. One female of the low dose group showed vaginal hemorrhage towards or after the end of the gestation period (days 23 - 26 pc), and was not able to deliver the pups, which were palpable earlier in the abdomen of this dam. After day 26 pc, no pups could be palpated for this dam.
 
- There were no substance-related clinical findings in the F1 dams during the lactation of F2 litters. Only one dam of the low dose group and one dam of the high dose group did not nurse their pups properly; all pups of high dose dam were cannibalized and/or died intercurrently. Furthermore, another low dose dam showed blood in bedding during the first days of the lactation period, and was not able to deliver its litter completely. It delivered only 2 pups which were cannibalized on day 1 pp.

FOOD CONSUMPTION
The mean food consumption of the males and females of 5000 ppm test group was clearly reduced during the premating period, the differences in comparison to the controls being statistically significant at most time intervals. In total, the high dose males consumed about 9% and the females about 8% less food than the respective control animals during the premating phase. Food intake was also statistically significantly diminished in the females of this test group (5000 ppm) during the gestation period (days 7-20 pc) and during the lactation period (days 7-14 pp only). The reduction in food consumption of the 5000 ppm males and females was considered to be related to the administration of the test substance. All other differences in food consumption between the groups are without any biological relevance.
 
WATER CONSUMPTION
In comparison to the respective control values the water consumption of the 2500 and 5000 ppm F1 males and females was distinctly lower during the premating period, the differences being statistically significant at all intervals in the high dose level and in several but not all intervals at the intermediate dose. In total a clear dose-response relationship was observed: high dose males consumed about 18%, intermediate dose males about 9% less water than control males; for high dose females about 27% and for intermediate dose females about 13% less water intake than in the female controls was recorded. Water consumption was also reduced during gestation and lactation periods in these test groups, again more pronounced in the high than in the 2500 ppm group. The water consumption of the animals of the low dose group (500 ppm) reached or even exceeded the relevant control values during premating, gestation and the lactation periods. The distinct reductions in the drinking water consumption in both sexes at 2500 and 5000 ppm are considered treatment-related, whereas the differences in water consumption between the low dose group and the control are considered to be without toxicological relevance.
 
BODY WEIGHT
In the F1 males, statistically significant reductions in mean body weights were seen in the highest dose group (5000 ppm) throughout the total study period (about 87% of the control value at the end of this study interval). Body weight gains of the 5000 ppm males, however, were generally similar to the respective control values. In total, the weight gain of the high dose F1 males was only about 5% lower than the body weight gain of the control males. Body weights of the 5000 ppm females were also statistically significantly reduced during the premating period (about 89% of the control value at the end of the premating phase). During gestation and lactation of F2 litter, mean body weights of the high dose F1 dams were statistically significantly lower than the corresponding control values. During premating, gestation and lactation periods body weight gain of the high dose females reached or even exceeded body weight gain of the controls.
The statistically significantly lower body weights recorded for the 5000 ppm F1 males and females were considered to be related to the test substance administration. A lower body weight was also recorded for these animals at the pup stage; during the following premating period the F1 parental animals of the high dose group gained substantially as much weight as the controls, but the body weights of the 5000 ppm rats were still reduced. All differences between the controls and 500 or 2500 ppm groups concerning body weights/weight gains, however, were regarded as spontaneous in nature.
 
MALE REPRODUCTIVE FUNCTION
For all F1 males which were placed with females to generate F2 pups, mating was confirmed. The male mating index was 100% in all groups.
 
FEMALE REPRODUCTIVE FUNCTION
The female mating index reached 100% in all groups. The mean duration until sperm was detected (day 0 pc) varied between 2.1 and 3.2 days and was highest for the control group, because one dam of this group had a prolonged cohabitation time. In the scheduled mating interval (F2), 2 control females did not become pregnant. Therefore, the fertility index was lowest in the control group (92%), whereas it was 100% in all substance-treated groups. There were no biologically relevant differences between test groups and the controls concerning the mean duration of gestation and the number of liveborn and stillborn F2 pups. All pregnant females - except one low dose female which had palpable pups in the uterus but did not deliver - gave birth to litters with liveborn pups. Consequently, the gestation and the live birth indices were not influenced by the administration of the test substance. The mean number of delivered pups/dam was not influenced by the test substance administered.

GROSS AND HISTOPATHOLOGICAL FINDINGS
- Thickening of the limiting ridge (margo plicatus) of the forestomach in most male and female rats.
- Minimal hyperkeratosis at the limiting ridge of the forestomach in most male and all female rats.
- Edema in the submucosa of the glandular stomach of 2 male and 10 female rats, minimal in all cases.

Dose descriptor:

NOAEL

Effect level:

240 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: general toxicity

Dose descriptor:

NOAEL

Effect level:

460 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: fertility

Remarks on result:

other: Generation: P and F1 (migrated information)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

**** F1 GENERATION PUPS/LITTERS ****

VIABILITY
No substance-induced effects on pup mortality/viability were recorded during the lactation period. Both the viability index, as an indicator of the viability of the pups during the first 4 days after birth, and the lactation index, as an indicator how the pups were nursed during the rest of their rearing, do not show differences of biological relevance.
 
SEX RATIO
The sex distribution and sex ratios of live F1 pups on the day of birth and on day 21 post parturition (pp) did not show any substantial difference between controls and treated groups; all differences observed are regarded as spontaneous.
 
BODY WEIGHT
Mean body weights of F1 male and female pups were clearly reduced in 5000 ppm test group from day 14 pp onwards and impaired in the intermediate dose (2500 ppm) on day 21 pp when compared to the controls. On day 21 pp the pup weights (both sexes combined) in the high dose group were about 35% and those of the 2500 ppm pups about 11% lower than the corresponding control values. Body weight gains of the 2500 ppm and 5000 ppm pups were also statistically significantly decreased from days 7 (5000 ppm) or 14 pp (2500 ppm) up to weaning (day 21 pp). The reductions in pup body weights/body weight gains in the 5000 and 2500 ppm groups were attributed to the test substance administration. All other differences concerning pup body weights/body weight gains are without any biological relevance and lie within the biological range of variation.
 
CLINICAL OBSERVATIONS
- None of the F1 pups of any one group showed abnormal clinical findings during the lactation period.
- There were no biologically relevant differences between the control and the substance-treated F1 pups in the several morphological development stages monitored up to weaning.
- No remarkable differences between the groups were observed in the different behavioral tests which the pups underwent up to weaning.
- Only spontaneous findings were seen at necropsy (e.g. incisors sloped, hernia diaphragmatica, dilated renal pelvis) in very few of the pups examined. All findings were present in the concurrent control at a comparable frequency and/or did not show a clear relation to dosing.



**** F2 GENERATION PUPS/LITTERS ****

PUP NUMBER AND STATUS
The mean number of delivered F2 pups/dam in the treatment groups was similar to the relevant control value; moreover, the percentages of liveborn and stillborn F2 pups were comparable between the groups; all differences between the groups are in the range of biological variation.
 
VIABILITY/MORTALITY
During the lactation period a statistically significant increase in pups cannibalized by dams was noted in the high and intermediate dose groups. The increased cannibalization rate was predominantly caused by just one intermediate dose dam and two high dose dams. One Female of the high group, however, neglected her pups during the lactation period, thus nearly all pups of this dam died before schedule and/or were cannibalized. Occasionally insufficient nursing behavior and cannibalism occured also in control females, and thus the higher rate of cannibalized pups at these dose levels was not considered treatment-related.
There were no differences in biological relevance between the control and the 500, 2500 and 5000 ppm F2 pups concerning viability and mortality; consequently the viability and lactation indices were not affected by the test substance administration (although some statistically significant differences existed). All relevant values are inside the historical control range and/or do not show a clear relation to dosing; moreover it had to be taken into consideration, that the high dose dams delivered on average distinctly more pups (14.0 pups/dam) than the controls (12.9 pups/dam).
 
SEX RATIO
No remarkable differences between the control and the substance-treated groups were found in respect to the sex ratio of the F2 pups. The observable differences are in the range of biological variation.
 
BODY WEIGHT
Mean body weights/body weight gains of the F2 male and female pups of the 5000 ppm and 2500 ppm groups were clearly influenced by the test substance administration. Mean pup body weights of the 5000 ppm pups were statistically significantly lower than the corresponding control values from days 14 (males and females) until weaning on day 21 pp, when the high dose pups (both sexes combined) weighed about 32% less than the controls. Mean pup body weights of the 2500 ppm pups were statistically significantly (about 12%) lower than the corresponding control values on day 21 pp (both sexes combined). Weight gains of the pups of the 2500 and 5000 ppm test groups were also statistically significantly reduced from the second week of the lactation period onward, the reduction more pronounced in the 5000 ppm than in the 2500 ppm pups. All differences between the control group and the 500 ppm group concerning pup body weight data of the F2 generation were considered spontaneous in nature.
 
CLINICAL OBSERVATIONS
- F2 generation pups did not show any clinical signs up to weaning which could be attributed to the treatment. Hydrocephaly, which occurs also occasionally in control pups was recorded in one 500 ppm pup.
- Development stages: There was a statistically significantly lower incidence of F2 pups/litter with auditory canal opening on time in the intermediate dose group and with eye opening on time in the 5000 ppm group. The relevant values were within the historical control ranges and a clear relation to dosing was not observed. These effects must be considered in conjunction with the retarded weight gain of these pups and were therefore assessed as being possibly substance-related. There were no differences of biological relevance in different stages of development between the low dose and the control pups.
- No substantial differences could be noted between the F2 pups of all test groups and the control pups in the different behavioral tests. The observable differences were without biological relevance.
- The examinations of F2 pups at necropsy did not reveal any differences considered to be of biological relevance between the controls and the substance-treated groups either in the type or in the number of pup necropsy observations. A few pups of the different groups showed some spontaneous findings like hernia diaphragmatica, incisors sloped, dilated renal pelvis, hydroureter, hydrocephaly, focal liver necrosis, cardiomegaly, septal defect and post mortem autolysis.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

53 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: general toxicity

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

53 mg/kg bw/day

Sex:

male/female

Basis for effect level:

other: general toxicity

Key result

Reproductive effects observed:

not specified

Mean Body Weight Changes (F0 parental animals), grams

Treatment week	0 mg/kg bw/d		53 mg/kg bw/d		240 mg/kg bw/d		460 mg/kg bw/d
	male	female	male	female	male	female	male	female
0 - 1	52.6	23.8	52.2	24.8	52.3	23.6	46.7^**	23.1
1 - 2	53.7	22.1	55.5	22.0	54.4	22.3	51.6	21.8
2 - 3	47.2	16.2	45.8	17.2	46.9	17.5	44.8	14.6
3 – 4	35.4	12.9	34.8	17.1^*	35.1	14.9	32.4	17.8^*
4 – 5	29.1	15.7	27.6	13.3	28.6	12.6	27.0	13.7
5 – 6	21.0	9.4	21.8	8.8	23.3	12.5	23.2	11.0
6 – 7	23.2	8.5	24.7	11.7	21.2	10.1	19.3^*	10.7
7 – 8	20.4	7.7	17.7	10.3	19.8	7.8	18.5	8.6
8 – 9	19.3	9.8	19.1	7.1	18.0	7.2	16.6	8.1
9 – 10	13.1	4.7	13.0	4.5	14.6	7.9^*	11.9	6.0
10 – 11	-7.0		-2.8		-2.8		-2.8
11 – 12	21.8		18.5		18.4		14.7^**
12 – 13	14.8		13.3		13.7		11.1
13 – 14	8.0		10.7		9.7		6.1
14 – 15	9.2		7.0		7.5		8.1
15 – 16	10.5		10.4		8.0		9.9
16 – 17	5.7		7.9		7.3		4.6
17 – 18	2.7		4.6		1.7		1.4
18 – 19	1.1		0.2		1.3		1.6
19 - 20	4.1		6.7		5.8		5.0

^*P<0.05

^**P<0.01

Reproduction and litter data for F0 parents /F1 pups

	0 ppm	500 ppm	2500 ppm	5000 ppm
Parents
Females mated	25	25	25	25
Females pregnant	24	23	23	24
Females with delivery	24	23	23	24
Mean duration of gestation (d)	22.0	21.9	21.9	21.9
Litter means
Live births/litter	13.8	13.8	13.7	14.3
Survivors day 4 preculling	13.3	13.5	13.4	13.8
Survivors day 4 postculling	7.5	7.9	7.9	7.9
Survivors day 21	7.5	7.9	7.8	7.8
Weight at day 1 (g) M/F	6.4/6.1	6.6/6.2	6.5/6.2	6.5/6.2
Weight at weaning (g) M/F	52.3/50.01	52.1/49.4	46.6^**/44.6^**	34.2^**/32.7^**
Sex ratio of live newborns % M/F	51/49	49/51	55/45	53/47
Selected as parents for the next generation M/F	25/25	25/25	25/25	25/25

^**P≤0.01

Reproduction and litter data for F1 parents /F2 pups

	0 ppm	500 ppm	2500 ppm	5000 ppm
Parents
Females mated	25	25	25	25
Females pregnant	23	23	23	24
Females with delivery	23	23	23	24
Mean duration of gestation (d)	22.0	21.9	21.9	21.9
Litter means
Live births/litter	12.5	11.6	12.0	13.8
Survivors day 4 preculling	11.8	10.6	10.8	12.5
Survivors day 4 postculling	7.7	7.0	7.5	7.8
Survivors day 21	7.7	6.9	7.5	7.4
Weight at day 1 (g) M/F	6.3/5.9	6.5/6.2	6.1/5.8	6.4/6.1
Weight at weaning (g) M/F	50.4/48.4	52.1/49.4	44.6^**/42.4^**	34.5^**/33.2^**
Sex ratio of live newborns % M/F	47/53	55/45	53/47	49/51

^**P≤0.01

Development landmarks in the F2 (mean % of pups reaching criteria/litter)

Parameter	0 ppm	500 ppm	2500 ppm	5000 ppm	Historical control range
Pinna unfolding	93.6 (16.65)	93.5 (20.87)	80.7 (33.37)	86.9 (26.87)	74-100
Auditory canal opening	98.8 (3.87)	95.1 (20.90)	91.4^*(18.09)	94.2 (12.26)	81-100
Eye opening	93.2 (18.08)	92.4 (21.89)	92.4 (21.89)	86.5^*(21.15)	85-100

^*P≤0.05

Figures in parentheses indicate standard deviations.

Conclusions:

Under the study conditions, acrylic acid had no adverse effects on reproductive parameters of the parental animals of either generation (F0 and F1) of all groups (500, 2500 and 5000 ppm). Therefore, the NOAEL with respect to reproductive function was 5000 ppm (equivalent to 1325 mg/kg/day zinc diacrylate). The NOAEL with respect to general toxicity of the test substance was 2500 ppm for the F0 generation parental animals (equivalent to 691 mg/kg/day zinc diacrylate) and 500 ppm for the F1 males and females and the offspring ( F1 and F2 pups) (equivalent to 153 mg/kg/day zinc diacrylate)

Executive summary:

A study was conducted to evaluate the reproductive toxicity potential of the test substance in rats for two generations. The aqueous acrylic acid solution was administrated at concentrations of 500, 2500 and 5000 ppm to F0 and F1 to parental animals over two-generation in reproduction toxicity. The continuous administration of aqueous acrylic acid solutions to rats over two generations caused clear signs of toxicity in the highest dose group (5000 ppm) in F0 and F1 parents. General toxicity was substantiated by e.g. reduced food and/or water consumption, impairment of body weights/body weight gains and gross and histopathological findings in the fore and the glandular stomach (i.e. thickening of and minimal hyperkeratosis at the limiting ridge (margo plicatus), edema in the submucosa of the glandular stomach), which are a consequence of the administration of the acid solutions. At 2500 ppm the water consumption of the F1 parental animals was still clearly reduced, but no further substance- related adverse effects on the parental rats were seen. Clear adverse substance-induced effects were also noted for the progeny of the high dose of the F0 and F1. Impaired body weight/body weight gain in the Fl and F2 pups and some indications for delays in the morphological development of the F2 pups were seen. The latter finding was likely associated with the decreased body weight/body weight gain. Similar, but much less pronounced effects were also observed for the F1 and/or F2 pups at 2500 ppm. 500 ppm were tolerated by both parental generations and their offspring without any changes which could be causally related to test substance administration. Under the study conditions, acrylic acid had no adverse effects on reproductive parameters of the parental animals of either generation (F0 and F1) of all groups (500, 2500 and 5000 ppm). Therefore, the NOAEL with respect to reproductive function was 5000 ppm (equivalent to 1325 mg/kg/day zinc diacrylate). The NOAEL with respect to general toxicity of the test substance was 2500 ppm for the F0 generation parental animals (equivalent to 691 mg/kg/day zinc diacrylate) and 500 ppm for the F1 males and females and the offspring ( F1 and F2 pups) (equivalent to 153 mg/kg/day zinc diacrylate) (BAMM, 1994).

Reference 3

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

not applicable

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague-Dawley Breeding Laboratories, Harlan Sprague-Dawley, Inc., Indianapolis, IN, USA
- Age at study initiation: 30-35 d
- Housing: Polycarbonate cages with stainless-steel wire lids
- Diet: Rodent chow(Lab Diet, Richmond Standard, PMI Feeds, Inc., St. Louis, MO), ad libitum
- Water: Deionized water, ad libitum
- Acclimation period: 2 wk


ENVIRONMENTAL CONDITIONS
- Temperature: 21.1 to 25.5 °C
- Humidity: 50-55%
- Air changes: 1/10 min
- Photoperiod : 12 h light/12 h dark cycle

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: 97% ZnCl2 was dissolved in milli-Q water.

Details on mating procedure:

- Length of cohabitation: 21 d
- Proof of pregnancy: Conception (day 0 of gestation)was checked daily in the mornings by looking for the presence or absence of copulatory plugs.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Not applicable

Duration of treatment / exposure:

2 generations

Frequency of treatment:

7 d/wk

Details on study schedule:

Dosing (7 days/week) started after two weeks of acclimation and was continued for males and females for 77 days prior to cohabitation. Dosing was continued throughout the periods of cohabitation (21 days) for both sexes. Dosing of female rats was continued throughout the gestation (21 days) and lactation (21 days) periods.
The doses for both sexes were adjusted weekly according to
changes in body weight.

Dose / conc.:

7.5 mg/kg bw/day (nominal)

Dose / conc.:

15 mg/kg bw/day (nominal)

Dose / conc.:

30 mg/kg bw/day (nominal)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dosage levels were derived from a 14-day dose range finding study. The maximum tolerated dose (MTD) of ZnCl2 was set at 60 mg/kg/day in rats. In order to prevent a large effect of zinc-induced toxicity on non-reproductive tissues interfering with the interpretation of pure reproductive toxicity, the high-dose group (group 4) was set at 1/2 (30.00 mg of ZnCl2/kg bw/d) of the established MTD. Likewise, the middose group (group 3) was at 1/4 (15.00 mg of ZnCl2/kg of bw/d) of the established MTD and the lowest dose group (group 2) was 1/8 (7.50 mg of ZnCl2/kg bw/d) of the established MTD.

Positive control:

No data

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily


DETAILED CLINICAL OBSERVATIONS: Yes


BODY WEIGHT: Yes


OTHER:
Hematology and clinical chemistry: Prior to necropsy, the Fo males were anesthetized with a combination of intraperitoneal Pentothal and Isoflo via inhalation. While the male rats were still under anesthesia, blood samples for hematology and clinical chemistries were collected in heparinised 3mL syringes via cardiac puncture. Following sample collection and while still under anesthesia, the animals were exsanguinated and necropsied. All plasma samples were analysed for alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALK), amylase (Amyl), blood urea nitrogen (BUN), creatinine (Crea), cholesterol (Chol), sodium (Na), potassium (K), chloride (Cl), calcium (Ca), phosphorus (Phos), albumin (ALB), total protein (TP), total bilirubin (Tbil), and glucose (Glu) using Roche Cobas Mira S Chemistry Analyser (Roche Diagnostic System, Inc., Somerville, NJ).

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

No data

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- Maximum of 8 pups/litter (4sex/litter); excess pups were killed and discarded.


PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring: Total litter size, number of stillborn pups per sex, sex distribution, pup body weight and the presence of any obvious external congenital anomalies


GROSS EXAMINATION OF DEAD PUPS:
No

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, as soon as possible after the last litters in each generation were produced
- Maternal animals: All surviving animals, after the last litter of each generation were weaned


HISTOPATHOLOGY / ORGAN WEIGHTS:

Organ weights: During the necropsy, organ weights were recorded for the kidneys, liver, brain, pituitary, adrenals, pancreas, thymus, spleen, testes, epididymides, prostate, and seminal vesicles. Fo male organ weights were also adjusted to body weight for statistical analysis.

Histopathology: Tissue samples collected from organs listed above for histopathologic evaluation were fixed in either Bouins solution (all reproductive tissues) or 10% neutral buffered formalin (all other tissues). After fixation, the tissue samples were trimmed, processed, embedded in paraffin, cut at 6 μm and stained with hematoxylin and eosin.

Postmortem examinations (offspring):

At the end of cohabitation for the parental F1 males and lactation for the F1 females, the animals were anesthesized, sacrificed and their organ weights
were recorded like their Fo parents.

Statistics:

- Kruskal-Wallis test followed by the Mann-Whitney U test for pair-wise comparisons to detect the difference between treatment group and control means
- ANOVA for analysing body-weight change, fertility, litter size, pups’ viability, pups’ body weight, postpartum dam weight and organ weight data between different treatment groups
- Dunnett’s and/or Duncan’s multiple comparison procedures

Reproductive indices:

The reproductive parameters were expressed in terms of indices, weights, ratios and efficiencies that considered all stages from conception to weaning. The parameters were:
- Fertility index (%) = (number of females delivering/number of females cohabited) × 100
- Live birth index (%) = (number of live pups at Day 0/number of pups born) × 100
- 4-d survival index (%) = (number of live pups on Day 4/number of pups alive on day 0) × 100
- Body weights of pups = the body weight of pups were recorded on days 0, 4, 7, 14 and 21
- Sex ratio (%) = (the total number of males on the day of weaning)/ (the total number of females on the day of weaning) × 100
- Food efficiency = (body weight gain/amount of diet consumed) × 100

Offspring viability indices:

- 21-d (weaning) survival index (%) = (number of pups alive on Day 21/number of pups alive on Day 4) × 100
- Litter Size = Number of pups/number of pregnant females

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

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS): Aggression/hyperactivity throughout the study in both males and females, hair loss behind the ears in males, vaginal discharges in low and high dose females; 0-20 and 12-24 % mortality in males and females respectively.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): All ZnCl2-treated F0 males experienced significant reduction in body weight after the 1st week of dosing and this trend continued up to the end of the experiment. The total weight gain of males was significantly reduced (dose dependent) in the low-, mid- and high-dose groups. The males experienced 0, 8, 20, and 12% mortality in control, low-, mid- and high-dose groups, respectively. In the F0 females, total weight gain and percent reduction in the low- mid- and high-dose groups were not significantly different from the control.


HEMATOLOGY AND CLINICAL CHEMISTRY: None of the hemogram or leukogram values of both Fo and F1 males and females among the ZnCl2-treated groups were different from those of the control groups. However, there was a trend toward decreased values of Packed Cell Volume (PCV). The clinical chemistry findings in males and females of both generations did not show any significant difference from those of their controls. However, in mid- and high-dose males of both generations, there seemed to a trend toward elevated values of Amyl, ALK, and GLu.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): In F0 rats, ZnCl2 treatment caused a significant reduction on the fertility, litter size, and the viability indices (Days 0 and 4) were significantly reduced at the high-dose group compared to control.

ORGAN WEIGHTS (PARENTAL ANIMALS): In F0 males, the unadjusted weights of the brain in the midand high-dose groups, the liver and kidney in all ZnCl2-treated groups, the spleen in the high-dose group, and the seminal vesicles in the mid- and high-dose groups were significantly different from the control.When organ weights of F0 males were adjusted for body weight, the brain in the mid- and high-dose groups, the liver and kidney in all ZnCl2-treated groups, the spleen in the high-dose group, and the seminal vesicles in the mid- and high-dose groups remained significantly different from their controls. The unadjusted organ weights of F0 females revealed significant differences for the spleen and uterus in the high-dose group. Following the adjustments of F0 female organ weights for body weight, the spleen and the uterus in the high-dose group remained significantly different from their controls.


GROSS PATHOLOGY (PARENTAL ANIMALS): Gross findings related to ZnCl2-treatment in males were primarily seen in the target organ systems (digestive, hematopoietic-lymphoreticular, and reproductive) already established for zinc. Digestive system lesions in the gastrointestinal tract (GIT) (distention, discoloration/hemorrhage and ulceration) and pancreas (smaller than usual) were mostly seen in rats given the two highest doses. Hematopoietic-lymphoreticular system lesions (small spleens and thymuses) were also scattered among the groups of ZnCl2-treated males. In the reproductive tract of the males, the only gross changes noted were small prostates and small seminal vesicles (one each) in the high-dose group. Gross lesions in ZnCl2-treated females generally paralleled those observed in their male counterparts.


HISTOPATHOLOGY (PARENTAL ANIMALS): In males, the most biologically meaningful lesions were found in the reproductive system (prostatic acinar atrophy and inflammation) and the hematopoietic-lymphoreticular system (splenic lymphoid depletion and hemosiderosis and thymic atrophy) of ZnCl2-treated groups. No significant changes in clinical pathology values or organ weights correlated with these lesions. None of the microscopic changes in target organs were of great magnitude. All unscheduled deaths were confined to the ZnCl2-treated groups, the majority of them probably being related to toxicity, but histomorphologic confirmation of this was not noted. The histopathology observed among the ZnCl2-treated females was similar to that seen in the males, except that no lesions were seen in the reproductive system. The correlations and biological interpretations were also very similar.


OTHER FINDINGS (PARENTAL ANIMALS):
Postpartum dam body weight: The F0 and F1 post-partum dam weights in all dose groups were significantly different from their control groups.

Key result

Dose descriptor:

NOAEL

Effect level:

7.5 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

haematology

organ weights and organ / body weight ratios

gross pathology

reproductive performance

Clinical signs:

effects observed, treatment-related

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:

effects observed, treatment-related

Histopathological findings:

effects observed, treatment-related

VIABILITY (OFFSPRING): The F1 males in the mid- and high-dose groups experienced a significant reduction in body weight after the 1st week of dosing and the low-dose group experienced a similar reduction after the 2nd week of dosing. These trends continued up to the end of the experiment. The total weight gain of F1 males was significantly reduced (dose dependent) in the low, mid-, and high-dose groups. The males experienced 0, 12, 8, and 4% mortality in the control, low-, mid- and high-dose groups, respectively. The mortality among the F1 females was 0, 8, 12, and 20% in the control, low-, mid- and high-dose groups, respectively.


CLINICAL SIGNS (OFFSPRING): Aggression/hyperactivity was observed throughout the study in both F1 males and females of ZnCl2-treated groups.


BODY WEIGHT (OFFSPRING): The body weights of F1 and F2 pups at Day 21 in the high-dose group were significantly lower compared to their control.


ORGAN WEIGHTS (OFFSPRING): In F1 males, the unadjusted weights of the brain, spleen, and prostate in all ZnCl2-treated groups, the liver, adrenal,
testis and seminal vesicles in mid-dose and the kidney in high-dose were significantly different from their controls. When the organ weights of F1 males were adjusted for body weight, the brain, spleen, and prostate in all ZnCl2-treated groups, the liver, adrenal and seminal vesicles in mid-dose group, and kidney in high-dose group remained significantly different from their controls. The unadjusted organ weights of F1 females that were different from their controls included the brain and spleen in low- mid- and high-dose groups and the kidneys in the high-dose group. Following the adjustments of F1 female organ weights for body weight, the brain and spleen in all dose groups and kidneys in high dose groups were significantly different from controls.


GROSS PATHOLOGY (OFFSPRING): Gross findings related to ZnCl2-treatment in males were primarily seen in the target organ systems (digestive, hematopoietic-lymphoreticular, and reproductive) already established for zinc. Digestive system lesions in the gastrointestinal tract (GIT) (distention, discoloration/hemorrhage and ulceration) and pancreas (smaller than usual) were mostly seen in rats given the two highest doses. Hematopoietic-lymphoreticular system lesions (small spleens and thymuses) were also scattered among the groups of ZnCl2-treated males. In the reproductive tract of the males, the only gross changes noted were small prostates and small seminal vesicles (one each) in the high-dose group. Gross lesions in ZnCl2-treated females generally paralleled those observed in their male counterparts.


HISTOPATHOLOGY (OFFSPRING): In males, the most biologically meaningful lesions were found in the reproductive system (prostatic acinar atrophy and inflammation) and the hematopoietic-lymphoreticular system (splenic lymphoid depletion and hemosiderosis and thymic atrophy) of 30.00 mg/kg/day ZnCl2-treated groups. These results indicated that ZnCl2 exposure has only mild effects on the reproductive performance of rats.

No significant changes in clinical pathology values or organ weights correlated with these lesions. None of the microscopic changes in target organs were of great magnitude. All unscheduled deaths were confined to the ZnCl2-treated groups, the majority of them probably being related to toxicity, but histomorphologic confirmation of this was not noted. The histopathology observed among the ZnCl2-treated females was similar to that seen in the males, except that no lesions were seen in the reproductive system. The correlations and biological interpretations were also very similar.


OTHER FINDINGS (OFFSPRING): Reproductive performance: F1: No significant difference was seen in the weaning index and sex ratios in F1 pups. In F1 generation rats, ZnCl2 treatment resulted in a significant reduction on fertility, viability (Day 0) and litter size in the high-dose group compared to control. However, ZnCl2 treatment showed no effect on viability index, weaning index and sex ratios of F2 pups.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

7.5 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: equivalent to ca. 4 mg Zn/kg bw/day and 11 mg zinc diacrylate/kg bw/day

Reproductive effects observed:

not specified

None

Conclusions:

Under the test conditions, administration of the test substance to adult male and female rats throughout maturation, mating, gestation and early lactation resulted in significant effects on adults and offspring at 30 and 15 mg/kg bw/day. Although effects were seen at 7.5 mg/kg bw/day, these were considered to be toxicologically non-significant and therefore this dose was considered to be the NOAEL.

Executive summary:

A study was conducted to evaluate the reproductive toxicity potential of test substance in rats for two generations. Male and female rats were administered test material at the doses of 7.50, 15.00 and 30.00 mg/kg bw/day over two successive generations. Control group animals received deionised water. Exposure of F0 and F1 parental rats to test material showed significant reduction in pups from the high-dose group but caused no effects on litter size, weaning index and sex ratio. There was significant reduction in body weights of F0 and F1 parental males and postpartum dam weights female rats. Exposure of test material to F0 and F1 generation parental animals resulted in non-significant change in clinical pathology parameters (except the ALK level). Reduction of brain, liver, kidney, spleen and seminal vesicles weights of males and in the spleen and uterus of females was observed in F0 and F1 rats. Gross lesions were observed in gastro-intestinal (GI) tract, lymphoreticular/ hematopoietic and reproductive tract in parental rats in both generations. Reduced body fat was also recorded in F1 parental rats. Under the test conditions, administration of the test substance to adult male and female rats throughout maturation, mating, gestation and early lactation resulted in significant effects on adults and offspring at 30 and 15 mg/kg bw/day. Although effects were seen at 7.5 mg/kg bw/day ( equivalent to  ca. 4 mg Zn/kg bw/day and 11 mg zinc diacrylate/kg bw/day), these were considered to be toxicologically non-significant and therefore this dose was considered to be the NOAEL (Khan, 2007).

Reference 4

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Males rats were fed diet supplemented with the test material for specific period and then mated with normal females. Males were sacrificed after mating and effect on sperm motility/viability and zinc concentration in reproductive organs observed. Females were allowed to have full term gestation and effect on conception and litter viability were determined.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: Charles-Foster

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: (P) Males: 162 g
- Diet: Crushed rat feed of Hindustan Lever (India); ad libitum

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Crushed rat feed

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 1 d
- Proof of pregnancy: Sperm in vaginal smear (mating performed only once irrespective of results)

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

30-32 d before mating to males only

Frequency of treatment:

Daily, ad libitum

Details on study schedule:

Following the day of mating, males were sacrificed and sperm was immediately collected from epididymis for motility and viability studies. Reproductive organs were dissected out for estimation of zinc. Mated females were allowed to have full term gestation.

Remarks:

Doses / Concentrations:
4,000 ppm zinc as zinc sulphate
Basis:
nominal in diet

No. of animals per sex per dose:

Control group: 15
Treatment group: 18

Control animals:

yes, plain diet

Details on study design:

None

Positive control:

None

Parental animals: Observations and examinations:

No data

Oestrous cyclicity (parental animals):

No data

Sperm parameters (parental animals):

Parameters examined in male parents:
Sperm motility and viability

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Number of pups, stillbirths, live births and malformation

Postmortem examinations (parental animals):

- Male animals: All animals sacrificed following the day of mating and reproductive organs (testis, epididymis, seminal vesicle and prostate) were dissected out for estimation of zinc
- Maternal animals: Not sacrificed

Postmortem examinations (offspring):

No data

Statistics:

Fischer's one sided T test

Reproductive indices:

None

Offspring viability indices:

None

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

effects observed, treatment-related

Reproductive performance:

effects observed, treatment-related

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS): Motility of the sperm was significantly reduced in the zinc treated rats at all time intervals viz. 30 min., 2 h and 4 h. The percentage reduction as compared to the controls was 8.5, 25 .3 and 29 .0 respectively. Sperm viability was unaffected. (See Table 3 for details)

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): Mating by zinc treated males caused significant lowering of incidence of conception and number of live births per mated female. Only 11 females conceived out of 18 mated by zinc treated males whereas all females mated with control rats conceived. (See Table 1 for details)

OTHER FINDINGS (PARENTAL ANIMALS): Zinc content was significantly increased only in the testis (25 %) and sperm (18 %) of the zinc treated rats. (See Table 2 for details)

Key result

Dose descriptor:

dose level: 4,000 ppm zinc

Sex:

male

Basis for effect level:

other: overall effects sperm characterization; other: zinc concentration in reproductive organs

Remarks on result:

not measured/tested

Remarks:

Effect level not specified (migrated information)

Key result

Dose descriptor:

other: Not treated but mated with males fed 4,000 ppm zinc

Sex:

female

Basis for effect level:

other: Overall effects incidence of conception

Remarks on result:

not measured/tested

Remarks:

Effect level not specified (migrated information)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

not specified

Behaviour (functional findings):

not specified

Developmental immunotoxicity:

not specified

VIABILITY (OFFSPRING): No stillbirth in any of the groups

OTHER FINDINGS (OFFSPRING): No malformed litter in any of the groups

Key result

Dose descriptor:

other: Offsprings of 4,000 ppm zinc treated males

Generation:

F1

Sex:

male/female

Basis for effect level:

other: overall effects live births; offspring malformation

Remarks on result:

not measured/tested

Remarks:

Effect level not specified (migrated information)

Key result

Reproductive effects observed:

not specified

Table 1: Outcome of mating by zinc treated male rats

	Mating males
	Control	Experimental
Number of females mated	15	18
Number of females conceived	15	11
Normal live litter
Total number	101	61
Number / mated female	6.73	3.38*

*Difference was significant at 5% level

Table 2: Zinc content of the reproductive tissues of zinc treated male rats

	Control	Experimental
Testis	28.1± 1.63 (15)	35.3 ± 2.13 (18)*
Epididymis	58.3 ± 3.46 (10)	61.4 ± 6.63 (10)
Seminal vesicle	24.4 ± 3.25 (10	33.8 ± 5.96 (10)
Prostate (whole)	48.8 ± 6.90 (10)	51.7 ± 6.36 (10)
Sperm	559 ± 14.8 (10)	658 ± 24.7 (10)*

Results expressed as Mean ± SEM in µg/g, except for sperm in µg/g dw, for the number of

rats shown in parentheses.

* P < 0.02

Table 3: Motility and viability of the sperm of zinc treated rats

	Control (15)	Experimental (18)
Motility (Emmen's unit)
30 min	25.9 ± 0.57	23.7 ± 0.47*
2 hours	23.7 ± 0.58	17.7 ± 1.15**
4 hours	17.2 ± 1.10	12.2 ± 1.42*
% viable at 4 hours	55.7 ± 4.80	49.0 ± 4.60

Values expressed as Mean ± SEM for the number of rats shown in parentheses.

* P < 0.01 

**P < 0.001

Conclusions:

Under the study conditions, dietary zinc supplementation at 4000 ppm reduced male fertility in rats.

Executive summary:

A study was conducted to determine the effects of dietary zinc supplementation on male fertility in Charles-Foster rats. 4000 ppm zinc as zinc sulphate was fed to 18 test males in diet for 30-32 d. 15 control males were fed normal diet for the same duration. All animals mated with individual normal females once between Day 30 and 32. After mating, males were sacrificed for sperm characterization and zinc concentration analysis in different reproductive organs. Mated females were allowed to have full term gestation. Mating by treated males caused significant lowering of incidence of conception and number of live births per mated female. However, no stillbirth or malformed litter was observed. Motility of the sperm was significantly reduced in the treated rats but viability was unaffected. Zinc content was significantly increased only in the testis and sperm of the treated rats. Under the study conditions, dietary zinc supplementation at 4000 ppm reduced male fertility in rats (Samanta, 1986).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

11 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Zinc sulphate studies

A study was conducted to determine the effects of dietary zinc supplementation on male fertility in Charles-Foster rats. 4000 ppm zinc as zinc sulphate was fed to 18 test males in diet for 30 -32 d. 15 control males were fed normal diet for the same duration. All animals mated with individual normal females once between Day 30 and 32. After mating, males were sacrificed for sperm characterization and zinc concentration analysis in different reproductive organs. Mated females were allowed to have full term gestation. Mating by treated males caused significant lowering of incidence of conception and number of live births per mated female. However, no stillbirth or malformed litter was observed. Motility of the sperm was significantly reduced in the treated rats but viability was unaffected. Zinc content was significantly increased only in the testis and sperm of the treated rats. Under the study conditions, dietary zinc supplementation at 4000 ppm reduced male fertility in rats (Samanta, 1986).

Zinc chloride studies

A study was conducted to evaluate the reproductive toxicity potential of test substance in rats for two generations. Male and female rats were administered test material at the doses of 7.50, 15.00 and 30.00 mg/kg bw/day over two successive generations. Control group animals received deionised water. Exposure of F0 and F1 parental rats to test material showed significant reduction in pups from the high-dose group but caused no effects on litter size, weaning index and sex ratio. There was significant reduction in body weights of F0 and F1 parental males and postpartum dam weights female rats. Exposure of test material to F0 and F1 generation parental animals resulted in non-significant change in clinical pathology parameters (except the ALK level). Reduction of brain, liver, kidney, spleen and seminal vesicles weights of males and in the spleen and uterus of females was observed in F0 and F1 rats. Gross lesions were observed in gastro-intestinal (GI) tract, lymphoreticular/ hematopoietic and reproductive tract in parental rats in both generations. Reduced body fat was also recorded in F1 parental rats. Under the test conditions, administration of the test substance to adult male and female rats throughout maturation, mating, gestation and early lactation resulted in significant effects on adults and offspring at 30 and 15 mg/kg bw/day. Although effects were seen at 7.5 mg/kg bw/day (equivalent to  ca. 4 mg Zn/kg bw/day and 11 mg zinc diacrylate/kg bw/day), these were considered to be toxicologically non-significant and is therefore this dose was considered to be the NOAEL (Khan, 2007).

Acrylic acid studies

Study 1:

A study was conducted to evaluate the reproductive toxicity potential of the test substance in rats for one generation. The method was equivalent to OECD Guideline415. 10 male and 20 female rats per group were administered acrylic acid at dose goals of 0, 83, 250 and 750 mg/kg/bw/day in drinking water. At the end of 13 weeks, the male and female rats from each group were mated one male to two females for a 15 d period. The F0 generation rats were sacrificed after weaning of the F1 generation and were approximately 194 d old at the time of sacrifice. The F1 generation rats were sacrificed at 21 d of age. Treatment effects were determined by statistical comparison of mortality, body weight change, food and water consumption, organ weight change and histological evaluation of tissues from sacrificed animals. Deleterious effects were observed at the high dose level in both sexes for parents and progeny. These effects included diet and water consumption, body and organ weight changes. Furthermore, there was a numerical reduction in the fertility and gestation indices and the number of pups weaned. At the intermediate level, although the reproductive parameters were equivalent to those of the controls, dose-related effects were seen for water consumption and body and organ weights in the parent rats. At the low dose level, organ weight changes observed in the female rats of the parent generation are considered to be of minimal biological significance. The results of the reproduction study do not indicate a substantial deleterious effect of acrylic acid on reproductive performance, since there were no statistically significant differences among the treated and control groups. However, the data should be interpreted cautiously because of a relatively atypical control group. For example, high dose females had relatively low fertility (45%) but this value was equivalent to that of the controls (50%). Although the average fertility index for earlier Fischer rat studies was 82%, the occurrence of a concurrent control group with lower fertility makes it impossible to determine whether the low fertility of the high dose females was treatment-related. Similarly, the typical (median) historical control value of 9 pups per litter for Fischer rats differs substantially from the control median of 6 in this study. Therefore, reduced litter size at the high dose level may or may not have been a treatment-related effect. In spite of the difficulty in interpreting the possible reproductive effects of acrylic acid at the high dose level, the results of the reproduction study indicate that there was no adverse effect at the two lower levels. This conclusion is justified whether one uses the concurrent control data or historical control data as basis for comparison. In summary, although numerous treatment-related effects were observed at the two highest dose levels, many of these may be the result of reduced food and water consumption rather than specific toxic effects of acrylic acid. At the highest dose level, the preponderance of statistical significance in many parameters in both parents and progeny concomitant with generally lowered reproductive indices is a clearly dose-related toxic effect. Therefore, based on the above findings the maximum dose level that did not produce a deleterious reproductive effect for one generation of exposure of acrylic acid in the drinking water of rats was estimated to be 250 mg/kg bw/day (equivalent to 1325 zinc diacrylate/kg/day) (IATG, 1980).

Study 2:

A study was conducted to evaluate the reproductive toxicity potential of the test substance in rats for two generations. The aqueous acrylic acid solution was administrated at concentrations of 500, 2500 and 5000 ppm corresponding to approx. 0, 53, 240 and 460 mg/kg bw/day)

to F0 and F1 to parental animals over two-generation in reproduction toxicity. The continuous administration of aqueous acrylic acid solutions to rats over two generations caused clear signs of toxicity in the highest dose group (5000 ppm) in F0 and F1 parents. General toxicity was substantiated by e.g. reduced food and/or water consumption, impairment of body weights/body weight gains and gross and histopathological findings in the fore and the glandular stomach (i.e. thickening of and minimal hyperkeratosis at the limiting ridge (margo plicatus), edema in the submucosa of the glandular stomach), which are a consequence of the administration of the acid solutions. At 2500 ppm the water consumption of the F1 parental animals was still clearly reduced, but no further substance- related adverse effects on the parental rats were seen. Clear adverse substance-induced effects were also noted for the progeny of the high dose of the F0 and F1. Impaired body weight/body weight gain in the Fl and F2 pups and some indications for delays in the morphological development of the F2 pups were seen. The latter finding was likely associated with the decreased body weight/body weight gain. Similar, but much less pronounced effects were also observed for the F1 and/or F2 pups at 2500 ppm. 500 ppm were tolerated by both parental generations and their offspring without any changes which could be causally related to test substance administration. Under the study conditions, acrylic acid had no adverse effects on reproductive parameters of the parental animals of either generation (F0 and F1) of all groups (500, 2500 and 5000 ppm). Therefore, the NOAEL with respect to reproductive function was 5000 ppm (equivalent to 1325 zinc diacrylate/kg/day). The NOAEL with respect to general toxicity of the test substance was 2500 ppm for the F0 generation parental animals and 500 ppm for the F1 males and females and the offspring ( F1 and F2 pups) (BAMM, 1994).

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

Justification for classification or non-classification

Based on studies conducted on substances representative of its individual constituents, the test substance does not warrant classification for reproductive toxicity according to EU CLP (1272/2008) criteria.