Dipotassium disulphate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment. This study has been performed in compliance with the: Swiss Ordinance relating to Good Laboratory Practice adopted May 18th, 2005 [SR 813.112.1]. This Ordinance is based on the OECD Principles of Good Laboratory Practice, as revised in 1997 and adopted on November 26th, 1997 by decision of the OECD Council [C (97)186/Final]. These principles are compatible with Good Laboratory Practice regulations specified by regulatory authorities throughout the European Community, the United States (EPA and FDA), and Japan (MHLW, MAFF and METI). There were no circumstances that may have affected the quality or integrity of the data.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

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
Animals: Rat, RccHan: WIST(SPF)
Rationale: Recognized by international guidelines as a recommended test system.
Breeder: Harlan Laboratories, B.V. Kreuzelweg 53 5961 NM Horst / Netherlands
Number of Animals: 40 males: 10 per group; 40 females: 10 per group
Age (at Start of Treatment): 11 weeks
Body Weight Range (at Start of Treatment): Males: 292 to 329 g; Females: 182 to 223 g
Identification: Cage card and individual animal number (ear tattoo).
Randomization: Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
Conditions: Standard laboratory conditions. Air-conditioned with 10-15 air changes per hour, continuously monitored environmental conditions (temp. range: 22±3 °C; relative humidity range: 30–73.5%). There was 12 hour fluorescent light / 12-hour dark cycle with music during the light period.
Accommodation: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding. During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular oestrus cycles.
Diet: Pelleted standard Kliba Nafag 3433 rodent maintenance diet was available ad libitum.
Water: Community tap-water from Füllinsdorf was available ad libitum in water bottles.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

DOSE FORMULATIONS: The dose formulations were prepared weekly using the test item as supplied by the Sponsor. Sodium sulphate was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using a magnetic stirrer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration.

STORAGE OF DOSE FORMULATIONS: Dose formulations were stored at room temperature (20±5 °C) in brown glass beakers.

TREATMENT
Method: Oral, by gavage
Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of studies.
Frequency of Administration: Once daily
Target Dose Levels: Group 1: 0 mg/kg/day (control group); Group 2: 100 mg/kg/day; Group 3: 300 mg/kg/day; Group 4: 1000 mg/kg/day
Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories Study C79092 (non-GLP), using dose levels of 100, 300 and 1000 mg/kg/day, resulting in a NOEL of 300 mg/kg/day.
Dose Volume: 10 mL/kg body weight
Duration of Acclimatization Period: 7 days
Duration of Treatment Period: Males (4 weeks); females (approximately 7 weeks)

Details on mating procedure:

MATING, GESTATION AND LACTATION

During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The females were removed and housed individually if the daily vaginal smear was sperm positive, or a copulation plug was observed.

The day of mating was designated day 0 post coitum.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSIS OF DOSE FORMULATIONS

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. Samples of about 2 g of each concentration were taken from the middle only to confirm stability (4 hrs and 7 days). During the second last week of the treatment, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were frozen (-20 ± 5 °C) and delivered on dry ice to Analytic Department (Harlan Laboratories Ltd., Itingen / Switzerland) and stored there at -20 ± 5 °C until analysis.

The samples were analysed by HPLC coupled to a conductivity detector following an analytical procedure developed at Harlan Laboratories. The test item was used as the analytical standard. Analysed samples were not discarded without written consent from the study director.

Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Füllinsdorf / Switzerland. The samples were not discarded without written consent from the study director.

The test item recoveries were found to be in the range of 86.7% to 97.4% with reference to the nominal concentration. The maximum variation from the mean of homogeneity samples which were taken from the top, middle and bottom of the samples, ranged from 0.5% to 4.7%. The stability of the application formulations was tested in samples taken four hours and seven days after preparation and stored at room temperature. The variation values ranged from 0.2% to 3.2% from the time-zero value.

Duration of treatment / exposure:

Males: 4 weeks
Females: Approximately 7 weeks

Frequency of treatment:

Once daily

Details on study schedule:

Acclimatization: 7 days (males and females)
First Test Item Administration: Day 1 of pre-pairing (males and females)
Pre-Pairing: 14 days (males and females)
Pairing: 11 days (males); 11 days maximum (females)
Gestation: Approximately 21 days (females)
Treatment Ends: On day before sacrifice (males); on day 3 post partum (females)
Necropsy: After a minimum of 28 days treatment (males); on day 4 post partum (females)

Remarks:

Doses / Concentrations:
100, 300, 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Parental animals: Observations and examinations:

Viability / Mortality: Twice daily
Clinical Signs: Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioural abnormalities in nesting and nursing.
Food Consumption: Males (weekly during pre-pairing and after pairing periods.); females (pre-pairing period days 1 - 8 and 8 - 14, gestation period days 0 - 7, 7 - 14 and 14 - 21 post coitum and lactation period days 1 - 4 post partum). No food consumption was recorded during the pairing period.
Body Weights: Recorded daily from treatment start to day of necropsy.

Litter observations:

The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 1 and 4 post partum.

Postmortem examinations (parental animals):

TERMINATION OF THE STUDY: Males were sacrificed after they had been treated for at least 28 days. Dams were sacrificed on day 4 post partum. When birth did not occur on the expected date (day 21 post coitum), the dam was sacrificed on day 25 post coitum.

NECROPSY: At the scheduled sacrifice, all parent animals were killed by an injection of sodium pentobarbital. All P generation animals were exsanguinated. All parent animals were examined macroscopically for any structural changes at the scheduled necropsy. For the parent animals, special attention was directed at the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulphide to visualize possible haemorrhagic areas of implantation sites.

ORGAN WEIGHTS: At the scheduled sacrifice, the testes and epididymides of all parental males were weighed separately.

TISSUE PRESERVATION: The ovaries from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution. The testes and epididymides from all parental males were preserved in Bouin’s fixative. The prostate and seminal vesicles from all males were fixed in neutral phosphate buffered 4% formaldehyde solution.

HISTOTECHNIQUE: All organ and tissue samples to be examined by the study pathologist were processed, embedded and cut at an approximate thickness of 2 - 4 micrometres and stained with haematoxylin and eosin. Additionally, the testes were stained by PAS-haematoxylin.

HISTOPATHOLOGY: Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the study pathologist. The same applied to all occurring gross lesions. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure. Histological examination of ovaries was carried out on any females that did not give birth. Microscopic examination of the reproductive organs of all infertile males was made. A histopathology peer review was performed by the Pathology Department (Harlan Laboratories Ltd., Itingen / Switzerland).

Postmortem examinations (offspring):

TERMINATION OF THE STUDY: Pups were sacrificed on day 4 post partum.

NECROPSY: At the scheduled sacrifice, all pups were killed by an injection of sodium pentobarbital. All pups were examined macroscopically for any structural changes at the scheduled necropsy.

Statistics:

The following statistical methods were used to analyse food consumption, body weights, reproduction data, macroscopical findings and organ weight:
- Means and standard deviations of various data were calculated.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test were applied if the variables could be dichotomized without loss of information.

Reproductive indices:

From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, post-implantation losses, mean litter size.

Offspring viability indices:

From the on-line recorded reproduction data, the following parameters were calculated: dead/live pups at first litter check, pup sex ratios and postnatal loss (up to day 4 post partum).

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

IN-LIVE DATA - PARENTAL ANIMALS

CLINICAL SIGNS OR OBSERVATIONS: All animals survived until the scheduled necropsy and no clinical signs were noted during the entire duration of the study.

FOOD CONSUMPTION OF MALES (Pre-pairing and After Pairing Periods): Mean food consumption was not affected by the treatment with the test item at any dose level. In order of ascending dose levels, the overall differences in food consumption were: ±0.0%, +2.4% and -0.8% during pre-pairing period and -1.7%, -7.4% and -2.9% during the after pairing period (percentages refer to the respective values of the control group).

FOOD CONSUMPTION OF FEMALES (Pre-pairing, Gestation and Lactation Periods): No statistically significant alterations of mean food consumption were observed at any dose level when compared to the respective values in the control group. In order of ascending dose levels, the overall differences in food consumption were: +6.0%, ±0.0% and -3.3% during the pre-pairing period, +1.6%, +2.0% and -3.6% during the gestation period and -3.7%, +4.0% and -5.6% during the lactation period (percentages refer to the respective values of the control group).

BODY WEIGHTS OF MALES (Pre-pairing, Pairing and After Pairing Periods): No significant changes were observed in mean body weight and mean body weight gain. In the order of ascending dose levels, the overall mean body weight gains were: +11%, +9%, +13% and +11% during the pre-pairing period, +6%, +5%, +5% and +4% during the pairing period and +4%, +4%, +4% and +3% during the after pairing period (percentages refer to the respective time intervals).

BODY WEIGHTS OF FEMALES (Pre-pairing, Gestation and Lactation Periods): No significant alterations of mean body weights and mean body weight gain were observed at any dose level when compared to the respective values in the control group. In the order of ascending dose levels, the overall mean body weight gain was +10%, +11%, +12% and +9% during the pre-pairing period and +57%, +59%, +57% and +57% during the gestation period and +5%, +4%, +6% and +6% during the lactation (percentages refer to the respective time intervals).


REPRODUCTION AND BREEDING DATA

MATING PERFORMANCE AND FERTILITY: The median and mean precoital times were unaffected by treatment with the test item. Mean precoital times were 3.3, 4.3, 3.3 and 3.1 days in order of ascending dose level. The median precoital time was 3, 4, 3 and 3 days in order of ascending dose level. Two females each in groups 1 and 2 were not pregnant. Thus the fertility indices were 80.0%, 80.0%, 100.0% and 100.0% in groups 1, 2, 3 and 4.

DURATION OF GESTATION: The mean duration of gestation was unaffected by treatment with the test item. Mean duration of gestation was 21.4, 21.5, 21.7 and 21.5 days, in order of ascending dose level.

CORPORA LUTEA COUNT: The mean number of corpora lutea per dam (determined at necropsy) was similar in all groups (14.6, 14.1, 14.2 and 14.1 in order of ascending dose level) and gave no indication of a test item-related effect.

IMPLANTATION RATE AND POST-IMPLANTATION LOSS: The mean number of implantations per dam and the post-implantation losses were unaffected by treatment with the test item. The mean numbers of implantations per litter were 13.3, 13.5, 14.0 and 13.3 in order of ascending dose level. The mean incidence of post-implantation loss as a percentage of total implantations was 10.4, 5.6, 10.0 and 3.8% in order of ascending dose level.

LITTER SIZE AT FIRST LITTER CHECK: The number of live pups at first litter check was unaffected by treatment with the test item. The mean number of live pups per litter was 11.9, 12.8, 12.6 and 12.8 in order of ascending dose level. The number of pups found dead at first litter check was two pups each in the control group and in group 2.

POSTNATAL LOSS DAYS 0 - 4 POST PARTUM: The total number of pup loss during the first four days was only one pup in the control group on day 3. Therefore the viability indices were 98.9% in the control group and 100.0%, in groups 2, 3 and 4.


TERMINAL FINDINGS - PARENTAL ANIMALS

ORGAN WEIGHTS: In males, weights (absolute and relative to body weight) of testes and epididymides were not affected by the treatment with the test item in any groups.

MACROSCOPICAL FINDINGS:

Males: In group 4, three males were noted to have the pelvis of right kidney dilated. In group 3, no abnormal findings were noted. In group 2, one male was noted to have both testes and epididymides reduced in size. In control group, one male was noted to have a pelvic dilation and enlargement of left kidney and another male to have both testes and epididymides reduced in size. Type and frequencies of these findings did not give an indication of a test item-related effect.

Females: No abnormal findings were noted in females.

HISTOPATHOLOGY FINDINGS: All findings recorded were within the range of normal background lesions which may be recorded in rats of this strain and age. In testes of animal no. 9 in group 1 and no. 19 in group 2, marked to severe tubular atrophy was found bilaterally and this finding was considered to be associated with their infertility. In the above 2 animals, aspermia and cellular debris in epididymides which were considered to be associated with the tubular atrophy of testes were also observed. No microscopic findings were found in other reproductive organs of these animals, reproductive organs of other infertile males (nos. 8 and 16) and ovaries of females (nos. 48, 49, 56 and 59) that did not give birth. Treatment with the test item did not reveal effects on the completeness of stages or cell populations. There was no indication for maturation arrest or any other degenerative type.

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: highest dose tested

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: highest dose tested

Remarks on result:

other: Generation: P (reproduction and development) (migrated information)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

LITTER DATA - F1 PUPS

EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION: At first litter check and during lactation, the clinical signs noted were wound on ear or on nose or on mouth in one pup in group 1, in four pups in group 3 and one pup in group 4 and hind foot injured and toes missing in one pup in group 2. None of these findings gave an indication of a test item-related effect.

SEX RATIOS: Sex ratios at first litter check and on day 4 post partum were unaffected by exposure to the test item. The proportion of males on day 4 post partum was 48%, 49%, 50% and 45% in order of ascending dose level.

PUP WEIGHTS TO DAY 4 POST PARTUM: Mean pup weights were unaffected by treatment with the test item. On day 1 post partum mean pup weights were 6.1, 6.1, 6.4 and 6.2 g for combined data of male and female pups in order of ascending dose level. Mean pup weight development during lactation was unaffected by treatment with the test item. Mean pup weights on day 4 post partum were 9.6, 9.4, 9.7 and 9.3 g for combined data of male and female pups in order of ascending dose level.

MACROSCOPICAL FINDINGS: No abnormal findings were noted at macroscopic examination of the pups.

Reproductive effects observed:

not specified

SUMMARY OF PERFORMANCE

P Animals Breeding for F1 Litters

 

Group (mg/kg/day)	1 (0)	2 (100)	3 (300)	4 (1000)
Female numbers	41-50	51-60	61-70	71-80
Number of females paired	10	10	10	10
Number of females mated	10	10	10	10
Number of pregnant females (A)	8	8	10	10
Number of females which reared their pups until day 4 post partum	8	8	10	10

(A)  Female Nos. 48 and 49 in group 1 and nos. 56 and 59 in group 2 were not pregnant.

Conclusions:

This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).

In absence of any effect the general NOEL (No Observed Effect Level) was established at 1000 mg/kg/day. As no effects were observed, the NOEL (No Observed Effect Level) for reproduction/ developmental toxicity was considered to be 1000 mg/kg/day.

Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of sodium sulphate on male and female reproductive performance such as gonadal function, mating behaviour, conception and parturition.

Four groups of 10 males and 10 females were treated by gavage with sodium sulphate once daily. Males were treated over a 14-day pre-pairing period and during the pairing period up to one day before necropsy. Females were treated throughout the pre-pairing, pairing, gestation and lactation period up to day 4 post partum.

 

The following dose levels were used:

                       Group 1:                0 mg/kg body weight/day (control group)

                       Group 2             100 mg/kg body weight/day

                       Group 3:            300 mg/kg body weight/day

                       Group 4           1000 mg/kg body weight/day

 

A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (highly purified water).

 

The following results were obtained:

In absence of any effect the general NOEL (No Observed Effect Level) was established at 1000 mg/kg/day. As no effects were observed, the NOEL (No Observed Effect Level) for reproduction/ developmental toxicity was considered to be 1000 mg/kg/day.

 

PARENTAL ANIMAL

 

General Tolerability

 

All animals survived until the scheduled necropsy and no clinical sign was observed during the study.

 

Food Consumption

 

Mean food consumption was not affected by treatment with the test item in males and females. 

 

Body Weights

 

No test item-related effects were observed in mean body weight and mean body weight gain of males and females.

 

Reproductive Data

 

Mating performance, fertility index and conception rate were not affected by treatment with the test item. 

 

The mean number of corpora lutea, the mean number of implantations per dam, and the post-implantation losses were unaffected by treatment with the test item. The mean duration of gestation was also unaffected by treatment with the test item.

 

Organ Weights

 

Mean weight of testes and epididymides were not affected by the treatment with the test item.

 

Macroscopical Findings and Histopathological Examinations

 

All organs and tissues examined did not reveal any macroscopic nor microscopic changes related to the treatment with the test item.  

 

LITTER DATA - F1 PUPS

 

The number of live pups at first litter check and the mean litter size was unaffected by treatment with the test item. Sex ratios at first litter check and on day 4 post partum were unaffected by treatment with the test item. Mean pup weights and weight gains were also not considered to be affected.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

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

No data were available for reproductive toxicity with the test substance. A reproduction/developmental toxicity screening study in rats with Na₂SO₄ was used as a read-across to fulfil the data gap for the test substance.The underlying hypothesis for the read-across between the test substance and Na₂SO₄ is the likelihood of common precursors and/or breakdown products, via physical or biological processes, which result in structurally similar chemicals (e.g. the metabolic pathway approach of examining related chemicals such as acid/ester/salt). Additional documentation, provided within the IUCLID Assessment Reports section, supports the read-across approach.

An OECD 421 reproduction/developmental toxicity screening study with Na₂SO₄ was evaluated in male and female rats after gavage administration at doses of 0, 100, 300, and 100 mg/kg/day for 4 weeks in males and to day 4 post partum in females (approximately 7 weeks). No test substance-related effects on mortality, clinical signs, body weight, food consumption, mating performance, fertility index, conception rate, corpora lutea, mean number of implants per dam, post-implantation losses, gestation length, gestation index, litter size, offspring survival, sex ratio, offspring body weight, organ weight (testes, epididymides) or macroscropic or microscopic were observed. Therefore, the NOAEL is 1000 mg/kg/day, the highest dose tested.

In addition, in 13-week and 2-year feeding studies with (NH₄)₂SO₄, no effects were reported on reproductive organs.

Short description of key information:
Based studies with the read-across chemical, Na2SO4, the test substance is not expected to be a reproductive toxin.

Justification for selection of Effect on fertility via oral route:
NOAEL is from a OECD Guideline, GLP study with the read-across chemical, Na2SO4.

Effects on developmental toxicity

Description of key information

Based studies with the read-across chemicals, H2SO4 and Na2SO4, the test substance is not expected to be a developmental toxin.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment. non-standard screening test.

Principles of method if other than guideline:

As part of a validation of a developmental screen, pregnant mice were exposed to 55 compounds, composed of known teratogens, known non-teratogens or equivocal substances.

GLP compliance:

no

Limit test:

yes

Species:

mouse

Strain:

other: ICR/SIM

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonsen Laboratories
- Age at study initiation: adult
- Weight at study initiation: 32 to 36 g
- Housing: housed one per cage in 32 X 23 X 15 cm suspended polycarbonate shoebox cages
- Diet (e.g. ad libitum): Simonsen Custom Lab Diet 7 ad libitum
- Water (e.g. ad libitum): UV-purified drinking water ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± ºC
- Photoperiod (hrs dark / hrs light): 12-hr-on, 12-hr-off light cycle

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Single daily dose by gavage; dose level at or near induction of maternal toxicity.

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: Mice were either received from the supplier timed-pregnant or (because of the low pregnancy rate in many shipments of these animals) were bred in-house.
- Proof of pregnancy: gestation day 1 = day plug found

Duration of treatment / exposure:

4 days (gestation day 8-12)

Frequency of treatment:

Once daily

Duration of test:

Up to day 22 of pregnancy

Remarks:

Doses / Concentrations:
2800 mg/kg/day
Basis:
actual ingested
test material

No. of animals per sex per dose:

28

Control animals:

yes, concurrent vehicle

Maternal examinations:

Maternal weight gain, delivery rate, litter size, % live births, pup weight on day 1 and day 3 were examined.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
- Examinations included: Dams that had not given birth by gestation day 21 or 22 were necropsied and their uteri were examined.

Fetal examinations:

Neonatal survival rate, macroscopic visceral and skeletal abnormalities were examined.

Statistics:

Maternal weight: two-tailed analysis of variance live and dead litter size: one-tailed analysis of variance neonatal survival rate: Fisher one-tailed exact probability.
Neonatal weight: two-tailed analysis of variance with litter size as co-variant.

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Normal maternal weight gain, normal delivery rate, normal litter size, normal number of live births.

Dose descriptor:

NOAEL

Effect level:

2 800 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

2 800 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Compared to controls, slight increase in neonatal body weight at day 1 p.p. (1.80+0.14 vs 1.72 + 0.13 grams). Normal weight of pups on day 3, no macroscopic visceral or skeletal abnormalities. The slight increase in body weight of neonates on day 1 p.p. only is not an adverse effect and is not biologically relevant.

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).
NOAEL (maternal toxicity) = 2800 mg/kg bw/ day
NOAEL (developmental toxicity) = 2800 mg/kg/bw/ day

Normal maternal weight gain, normal delivery rate, normal litter size, normal nr. of live births.
Compared to controls, slight increase in neonatal body weight at day 1 p.p. (1.80+0.14 vs 1.72 + 0.13 grams). Normal weight of pups on day 3, no macroscopic visceral or skeletal abnormalities.
The slight increase in body weight of neonates on day 1 p.p. only is not an adverse effect and is biologically totally irrelevant.

Executive summary:

The developmental effects of the test substance in the mouse were examined as part of a validation effort of a developmental screening test. The test substance was administered (2800 mg/kg/day) by gavage on gestation days 8 through 12. Females were allowed to deliver, and neonates were examined, counted, and weighed on the day of birth (day 1) and day 3. No mortality, an unchanged average weight gain, and normal number of litters and neonates/litter were found. No effect on maternal body weight was observed. A 100% perinatal survival was found, with an increased postnatal weight at day 1, and normal weight at day 3 in the absence of externally visible abnormalities.

Reference 2

Endpoint:

developmental toxicity

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Literature study, comparable to OECD 414 This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

number of exposure levels

GLP compliance:

not specified

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Langshaws Rabbitry, Augusta, Michigan
- Housing: wire-mesh cages
- Diet (e.g. ad libitum): given free access to commercial laboratory chow
- Water (e.g. ad libitum): yes
- Acclimation period: 3 weeks

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

whole body

Vehicle:

water

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Exposures were conducted under dynamic airflow conditions in 4.3 m3 stainless steel and glass Rochester-type chambers. The chamber airflow was approximately 800 L/min. The aerosol was generated for each chamber by nebulizing 2M H2S04 with a pneumatic atomizing nozzle. The concentration of H2S04 in the exposure chambers was analysed three times per day throughout exposure with a column made of DOWEX® 50 W x 8 with a water eluent using a conductivity cell detector. Particle size of the aerosol was determined on 4 different days of exposure using a particle size monitor. The mean of the average time-weighted daily concentrations of H2S04, the count median diameter of the H2S04 particles, and the average daily temperature and relative humidity in the chambers were recorded.

Analytical verification of doses or concentrations:

yes

Details on mating procedure:

Nulliparous female rabbits were artificially inseminated. The day on which rabbits were artificially inseminated was considered day zero of gestation

Duration of treatment / exposure:

Day 6-18 of gestation

Frequency of treatment:

7 hours/day

Duration of test:

Day 29 of gestation

Remarks:

Doses / Concentrations:
0, 5, 20 mg/m3
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
0, 5.7, 19.3 mg/m3
Basis:
analytical conc.

No. of animals per sex per dose:

20 females

Control animals:

yes

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on days 6, 9, 12, 15, 19 and 29 of gestation

POST-MORTEM EXAMINATIONS: Yes
- Organs examined: On day 29 of gestation, rabbits were sacrificed by carbon dioxide inhalation. The maternal liver was weighed. Respiratory tract tissues from 6 dams from each group were preserved in formalin and examined histologically. The nasal turbinates were decalcified and a section
taken through a transverse plane. Multiple sections of lung (minimum of 6 sections) and a transverse section of trachea were taken from each rabbit. The tissues were processed by routine histologic procedures and stained with haematoxylin and eosin prior to light microscopy.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included: The number and position of live, dead, and resorbed foetuses were noted. The uterus of each non-pregnant female was stained with a 10% solution of sodium sulphide stain and examined for evidence of implantation sites

Fetal examinations:

- External examinations: Yes: all per litter (All foetuses were weighed, measured (crown-rump length), sexed, and examined for external alterations
and cleft palate.)
- Soft tissue examinations: Yes: 1/3
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter

Statistics:

The Wilcoxon test as modified by Haseman and Hoel was used to evaluate the incidence of foetal alterations and resorptions; the litter was used as the experimental unit. Continuous data were analysed by a one-way analysis of variance and Dunnett's test. The level of significance chosen for all cases was p < 0.05.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
A statistically significant decrease in maternal body weight gain was noted during the first few days of exposure to 20 mg H2S04/m3. Histologic examination of the nasal turbinates, trachea, and lungs revealed a trend toward a dose-related increase in the incidence of subacute rhinitis and tracheitis; no changes in the lungs were seen which were considered to be due to exposure

Dose descriptor:

LOAEC

Remarks:

(rabbit)

Effect level:

19.3 mg/m³ air (analytical)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEC

Remarks:

(rabbit)

Effect level:

5.7 mg/m³ air (analytical)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The incidence of foetal malformations, considered individually or collectively, was not altered significantly by exposure. The only statistically significant evidence of embryotoxicity in rabbits was an increased incidence of small non-ossified areas in the skull bones (a minor variation in skeletal development) among the offspring of does exposed to 20 mg H2S04/m3.

Dose descriptor:

NOAEC

Remarks:

(rabbit)

Effect level:

19.3 mg/m³ air (analytical)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Dose descriptor:

NOAEC

Remarks:

(rabbit)

Effect level:

19.3 mg/m³ air (analytical)

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Conclusions:

No evidence of teratogenicity, embryotoxicity, foetotoxicity or developmental toxicity was seen under the conditions of this study, at exposure levels sufficient to cause mild maternal toxicity.
This study and the conclusions which are drawn from it fulfil the quality criteria (validity, reliability, repeatability).

Executive summary:

Inseminated female rabbits were exposed (whole body) to atmospheres containing aerosols of sulphuric acid at nominal concentrations of 0, 5, or 20 mg/m³ (measured concentrations of 0, 5.7, or 19.3 mg/m³, respectively) for 7 hours/day on Days 6 - 18 of gestation. Body weight and clinical signs were recorded. Animals were sacrificed at Day 29 of gestation, maternal liver was weighed, and respiratory tract tissues from 6 dams per group were preserved and examined histologically. The number and position of live, dead, and resorbed foetuses were noted. The sex, body weight, body length, and crown-rump length of each live foetus were recorded. External, skeletal, and visceral examinations were conducted. Evidence of mild toxicity was seen in maternal rabbits (reduced weight gain and respiratory tract effects) at 20 mg/m³. No evidence of teratogenicity or developmental toxicity was seen in rabbits.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 800 mg/kg bw/day

Species:

mouse

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

20 mg/m³

Species:

other: mice and rabbits

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

No data were available for developmental toxicity with the test substance. Inhalation studies in mice and rabbits with H₂SO₄ and an oral study in mice with Na₂SO₄ were used as a read across to fulfil the data gap for the developmental toxicity endpoint. The underlying hypothesis for the read-across between the test substance and H₂SO₄ and Na₂SO₄ is the likelihood of common precursors and/or breakdown products, via physical or biological processes, which result in structurally similar chemicals (e.g. the metabolic pathway approach of examining related chemicals such as acid/ester/salt). Additional documentation, provided within the IUCLID Assessment Reports section, supports the read-across approach.

Mated female mice were exposed (whole body) to atmospheres containing aerosols of H₂SO₄ at nominal concentrations of 0, 5, or 20 mg/m³ (measured concentrations of 0, 5.7, or 19.3 mg/m³, respectively) for 7 hours/day on Days 6 -15 of gestation. Body weight, clinical signs, food consumption, and water consumption were recorded. Animals were sacrificed at Day 18, maternal liver was weighed, and respiratory tract tissues from 6 dams per group were preserved and examined histologically. The number and position of live, dead, and resorbed foetuses were noted. The sex, body weight, body length, and crown-rump length of each live foetus were recorded. External, skeletal, and visceral examinations were conducted. Evidence of mild toxicity was seen in maternal mice (reduced food consumption, reduced liver weights) at 20 mg/m³. Gross and microscopic examination of the nasal turbinates, trachea, and lungs revealed no evidence of test substance-related toxicity. No evidence of teratogenicity or developmental toxicity was seen in mice.

Inseminated female rabbits were exposed (whole body) to atmospheres containing aerosols of H₂SO₄ at nominal concentrations of 0, 5, or 20 mg/m³ (measured concentrations of 0, 5.7, or 19.3 mg/m³, respectively) for 7 hours/day on Days 6 - 18 of gestation. Body weight and clinical signs were recorded. Animals were sacrificed at Day 29 of gestation, maternal liver was weighed, and respiratory tract tissues from 6 dams per group were preserved and examined histologically. The number and position of live, dead, and resorbed foetuses were noted. The sex, body weight, body length, and crown-rump length of each live foetus were recorded. External, skeletal, and visceral examinations were conducted. Evidence of mild toxicity was seen in maternal rabbits (reduced weight gain and respiratory tract effects) at 20 mg/m³. No evidence of teratogenicity or developmental toxicity was seen in rabbits.

The developmental effects of the test substance in the mouse were examined as part of a validation effort of a developmental screening test. The test substance was administered (2800 mg/kg/day) by gavage on gestation days 8 through 12. Females were allowed to deliver, and neonates were examined, counted, and weighed on the day of birth (day 1) and day 3. No mortality, an unchanged average weight gain, and normal number of litters and neonates/litter were found. No effect on maternal body weight was observed. A 100% perinatal survival was found, with an increased postnatal weight at day 1, and normal weight at day 3 in the absence of externally visible abnormalities.

Justification for selection of Effect on developmental toxicity: via oral route:
NOAEL is from scientifically valid study in mice with read-across chemical, Na2SO4

Justification for selection of Effect on developmental toxicity: via inhalation route:
NOAEL is from scientifically valid study in in mice and rabbits with read-across chemical, H2SO4

Justification for classification or non-classification

Based on the fact that no developmental toxicity was observed in mice and rabbits with the read across chemicals H₂SO₄ and Na₂SO₄, and no reproductive toxicity was observed with the read-across chemical Na₂SO₄, the test substance does not need to be classified for developmental or reproductive toxicity according the EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.