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REACH

Sulphur trioxide

EC number: 231-197-3 | CAS number: 7446-11-9

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproduction/Developmental Toxicity Screening Test according to OECD Test guideline 421: NOEL 1000 mg/kg bw/day (Ceccatelli, R. 2010); key study, reliability 1.

One generation reproductive study in the mouse: NOAEL 5000 ppm (eq. 3044.15 mg/kg bw/day) for reproductive and general toxicity (Andres, C.J. and Cline, T.R, 1989); WoE, reliability 3.

One generation reproductive study in the pig:: NOAEL 3000 ppm for reproductive and general toxicity (Paterson, D.W. et al. 1979); WoE, reliability 3.

Link to relevant study records

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start and completion dates: 22 April 2010 to 30 August 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

OECD guideline 421 (OECD guideline for testing of chemicals 421, “Reproduction/Developmental Toxicity Screening Test”, adapted by the Council on 27th July 1995.

Deviations:

GLP compliance:

yes (incl. QA statement)

Limit test:

Justification for study design:

To generate preliminary information using the OECD 421 "Reproduction/Developmental Toxicity Screening Test" concerning the effects of Sodium Sulphate on male and female reproductive performance such as gonadal function, mating behaviour, conception and parturition. Recognised by international guidelines as a recommended test system.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Supplied by the Sponsor. Treuhandgemeinschaft Deutscher Chemiefasererzeuger GmbH (TDC)
- Purity: 99.5%
- Expiry Date (Retest Date): 30 Sep 2010

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Application formulations were found to be homogeneously prepared with sufficient formulation stability under study storage conditions.
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: Soluble in highly purified water at formulated concentrations of 100, 300 & 1000 mg/kg bw/day. Stability of test formulations confirmed analytically.
- Reactivity of the test material with the incubation material used (e.g. plastic ware): None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): Formulated to concentration in purified water

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: None

Species:

rat

Strain:

Wistar

Remarks:

Rat, RccHan: WIST(SPF)

Details on species / strain selection:

Rat, RccHan: WIST(SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, B.V. Kreuzelweg 53, 5961 NM Horst, Netherlands.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 11 weeks
- Weight at study initiation: Males: 292 to 329 g, Females: 182 to 223 g
- Fasting period before study: No
- Housing: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J.Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst, Switzerland). During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (ad libitum): Pelleted standard Kliba Nafag 3433 rodent maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst, Switzerland (Batch No's. 83/09 and 22/10.
- Water (ad libitum): Community tap-water from Füllinsdorf was available in water bottles.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 30 - 73.5%
- Air changes (per hr): Air-conditioned with 10 - 15 air changes/hr
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle

IN-LIFE DATES: 29-Apr-2010 (test item administration) to 30-Aug-2010 (experimental completion)

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Purified water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dose formulations were prepared weekly. 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. On obtaining an homogeneous mixture, the remaining vehicle was added. Separate formulations were prepared for each concentration. Dose formulations were stored at room temperature in brown glass beakers.

VEHICLE
- Purified water
- Concentration in vehicle: 10, 20 and 100 mg/kg dosed at a dose volume of 10 mL/kg bw.
- Amount of vehicle (if gavage): Dose volume of 10 mL/kg with a daily adjustment to actual body weight.

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed.
- Proof of pregnancy: If the daily vaginal smear was sperm positive, or a copulation plug was observed. The day of mating was designated day 0 post coitum.
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Individually
- Any other deviations from standard protocol: No

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of Dose Formulations:
On treatment day 1, 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 (at 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 Dr. K. Morgenthal (Harlan Laboratories Ltd., Itingen, Switzerland) and stored there at -20 ± 5 °C until analysis. The samples were analyzed by HPLC coupled to a conductivity detector following an analytical procedure developed at Harlan Laboratories. The test item was used as the analytical standard. Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Füllinsdorf, Switzerland.

The linearity of the analytical systems used for sample analyses was demonstrated with a good
relationship between peak areas measured and working standard concentrations. All calibration
points used met the acceptance limit of ±20% variation from the calibration curve derived by
linear regression analysis. The regression coefficients calculated were found to be better than
0.99.

The Sodium Sulphate peak was assigned in sample chromatograms by comparison to that of
working standards. In blank sample chromatograms no peak appeared at the retention time of
Sodium Sulphate and, therefore, the absence of the test item in the control samples was
confirmed.

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.
Results indicate the accurate use of the test item Sodium Sulphate and purified
water as vehicle during this study. Application formulations were found to be homogeneously
prepared and sufficient formulation stability under storage conditions was approved.

Duration of treatment / exposure:

Duration of Treatment Period:
Males: 4 weeks
Females: ca. 7 weeks

Frequency of treatment:

Once daily with treatment ending on day 3 post partum for females and on the day before sacrifice for males.

Details on study schedule:

Study Sequence Males Females

Acclimatization 7 days 7 days
First Test Item Administration Day 1 of pre-pairing Day 1 of pre-pairing
Pre-Pairing 14 days 14 days
Pairing 11 days maximum 11 days
Gestation Approximately 21 day -
Treatment Ends On day 3 post partum On day before sacrifice
Necropsy On day 4 post partum After a min. of 28 days treatment

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control group

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Middle dose

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

40 males: 10 per group
40 females: 10 per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous non-GLP dose range-finding toxicity study in Han Wistar rats, Harlan Laboratories Study C79092, using dose levels of 100, 300 and 1000 mg/kg/day, resulting in a NOEL of 300 mg/kg/day.
- Dose selection rationale:
- Rationale for animal assignment: By computer-generated random algorithm.

Positive control:

Not applicable

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Viability / Mortality: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical signs: Once daily, during acclimatization and up to day of necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded daily from treatment start to day of necropsy.

FOOD CONSUMPTION (oral gavage study, not a feeding (diet) study:
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.

Oestrous cyclicity (parental animals):

During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.

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.

Sperm parameters (parental animals):

Parameters examined in [all/P/F1/F2] male parental generations:
[testis weight, epididymis weight, daily sperm production, sperm count in testes, sperm count in epididymides, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology, other:]

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
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.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Litter size, live births, sex ratio, still births and any gross anomalies. Pups were weighed individually (without identification) on days 1 and 4 post partum.

GROSS EXAMINATION OF DEAD PUPS: No.
ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No.
ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No.

Postmortem examinations (parental animals):

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 (nos. 48, 49, 56 and 59) that did not give birth. Microscopic examination of the reproductive organs of all infertile males (nos.
8, 9, 16 and 19) was made.

Postmortem examinations (offspring):

SACRIFICE
At the scheduled sacrifice, all animals were killed by an injection of sodium pentobarbital. Parental generation animals were exsanguinated.

GROSS NECROPSY
Pups were sacrificed on day 4 post partum. All pups were examined macroscopically for any structural changes at the scheduled necropsy.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not applicable for the litter (only parental animals).

Statistics:

The following statistical methods were used to analyze food consumption, body weights, reproduction data, macroscopical findings and organ weight:

• Means and standard deviations of various data were calculated.
• The Dunnett-test [see References (2)] (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 [see References (3)] (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 [see References (4)] were applied if the variables could be dichotomized without loss of information.

Reproductive indices:

Mating Performance and Fertility:
The median and mean precoital times were unaffected by test item treatment. 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 in groups 1 and two females in group 2 were not pregnant. Thus the fertility indices were 80.0%, (Group 1) 80.0% (Group 2), 100.0% (Group 3) and 100.0% (Group 4).

Fertility index = (Females achieving a pregnancy / Females paired) x 100

Offspring viability indices:

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.

Viability index = (number of alive pups on day 4 p.p. / number of pups born alive) x 100.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

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.
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.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

In the testes of animal no. 9 (Group 1) and no. 19 (Group 2), marked to severe tubular atrophy was found bilaterally and this finding was considered to be associated with their infertility. In both males, 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).

Treatment with the test item did not reaveal effects on the completeness of stages or cell
populations. There was no indication for maturation arrest or any other degenerative type.

Reproductive performance:

no effects observed

Description (incidence and severity):

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.

All animals survived until the scheduled necropsy and no clinical signs were noted during the
entire duration of the study.
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).
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).
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).
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).
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.
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.
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.
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.
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.
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.
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.
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.

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: An absence of any adverse effect, therefore the NOEL was established at 1000 mg/kg/day.

Key result

Critical effects observed:

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs at first litter check and during lactation 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.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The number of live pups at first litter check was unaffected by treatment with the test item. The number of pups found dead at first litter check was two pups each in the control group and in group 2.

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.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

not specified

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

Key result

Dose descriptor:

NOEL

Generation:

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No findings gave any indication of a test item-related effect.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

Key result

Reproductive effects observed:

Treatment related:

Table 1. Summary of food consumption in P0 male.

PRE-PAIRING PERIOD

Days 1-8 MEAN

ST.DEV. N

Days 8-14 MEAN

ST.DEV.

MEAN OF MEANS

Over PRE-PAIRING PERIOD

AFTER PAIRING PERIOD

Days 1-6 MEAN

ST.DEV. N

Group 1

mg/kg

24.8

1.0

25.7

1.6

25.3

24.2

Group 2

mg/kg

1.7

1.6

25.3

2.2 10

Group 3

mg/kg

1.5

2.0

25.9

2.8 10

Group 4

mg/kg

2.1

1.7

25.1

1.4 10

Table 2. Summary of food consuption P0 female

PRE-PAIRING PERIOD

Days 1-8 MEAN

ST.DEV. N

Days 8-14 MEAN

ST.DEV.

MEAN OF MEANS

Over PRE-PAIRING PERIOD

GESTATION PERIOD

Days 0-7 MEAN

ST.DEV. N

Days 7-14 MEAN

ST.DEV. N

Days 14-21 MEAN ST.DEV. N

Group 1

0 mg/kg

17.2

1.2

19.3

1.4

18.2

22.9

1.8

24.9

2.1

26.5

2.1 8

Group 2

100 mg/kg

18.2 1.4

20.3 1.2

19.3

22.6 4.0

25.6 1.4

27.6 1.5

25.2

31.1 2.7 8

Group 3

300 mg/kg

17.2 1.6

19.1 1.8

18.2

22.9 1.5

25.1 1.8

27.7 2.6

25.3

33.6 4.4 10

Group 4

1000 mg/kg

16.9 1.0

18.4 1.3

17.6

21.6 1.4

23.5 1.6

26.6 1.9

23.9

30.5 4.3 10

MEAN OF MEANS 24.8

Over GESTATION PERIOD (Days 0-21)

LACTATION PERIOD

Days 1-4 MEAN 32.3

ST.DEV. 5.5

N 8

*/**/- : DUNNETT-Test based on pooled variance sig. at 5% (*), 1% (**) or not sig. (-)

Table 3. summary of differences in mean food consumption of P0 males

PRE-PAIRING PERIOD

Days 1-8 MEAN

ST.DEV.

DIFF (%)*

Days 8-14 MEAN

ST.DEV.

DIFF (%)*

MEAN OF MEANS

Over PRE-PAIRING PERIOD

DIFF (%)*

Over PRE-PAIRING PERIOD

AFTER PAIRING PERIOD

Days 1-6 MEAN

ST.DEV.

DIFF (%)*

Group 1

mg/kg

24.8

1.0

---

25.7

1.6

---

24.2

1.1

---

Group 2

mg/kg

25.1

1.7

+1.2

25.4

1.6

-1.2

±0.0

23.8

2.2

-1.7

Group 3

mg/kg

25.7

1.5

+3.6

26.1

2.0

+1.6

+2.4

22.4

2.8

-7.4

Group 4

mg/kg

24.8

2.1

±0.0

25.4

1.7

-1.2

-0.8

23.5

1.4

-2.9

*: Percentage relative to the control group

Table 4. Summary of diffrenecs in mean food consumption in P0 Female

PRE-PAIRING PERIOD

Days 1-8 MEAN

ST.DEV.

DIFF (%)*

Days 8-14 MEAN

ST.DEV.

DIFF (%)*

MEAN OF MEANS

Over PRE-PAIRING PERIOD

DIFF (%)*

Over PRE-PAIRING PERIOD

GESTATION PERIOD

Days 0-7 MEAN

ST.DEV.

DIFF (%)*

Days 7-14 MEAN

ST.DEV.

DIFF (%)*

Days 14-21 MEAN ST.DEV.

DIFF (%)*

Group 1

mg/kg

17.2

1.2

---

19.3

1.4

---

22.9

1.8

---

24.9

2.1

---

26.5

2.1

---

Group 2

mg/kg

18.2

1.4

+5.8

20.3

1.2

+5.2

+6.0

22.6

4.0

-1.3

25.6

1.4

+2.8

27.6

1.5

+4.2

25.2

Group 3

mg/kg

17.2

1.6

±0.0

19.1

1.8

-1.0

±0.0

22.9

1.5

±0.0

25.1

1.8

+0.8

27.7

2.6

+4.5

25.3

Group 4

mg/kg

16.9

1.0

-1.7

18.4

1.3

-4.7

-3.3

21.6

1.4

-5.7

23.5

1.6

-5.6

26.6

1.9

+0.4

23.9

MEAN OF MEANS 24.8

*: Percentage relative to the control group

Table 5. Summary of P0 male body weight gain.

Pre-pairing period

Group 1 (0 mg/kg bw/day)

Group 2 (100 mg/kg bw/day)

Group 3 (300 mg/kg bw/day)

Group 4 ( 1000 mg/kg bw/day)

Day 1 MEAN ST.DEV.

Day 2 MEAN ST.DEV.

Day 3 MEAN ST.DEV.

Day 4 MEAN ST.DEV.

Day 5 MEAN ST.DEV.

Day 6 MEAN ST.DEV.

Day 7 MEAN ST.DEV.

Day 8 MEAN ST.DEV.

Day 9 MEAN ST.DEV.

Day 10 MEAN ST.DEV.

Day 11 MEAN ST.DEV. N

308 9

314 9

317 9

321

318

326

322

326

330

335

339

10 10

307 -

313 -

315 -

319 -

317 -

324 -

320 -

323 -

327 -

329 -

332 -

310 -

318 -

319 -

325 -

324 -

331 -

329 -

334 -

336 -

340 -

344 -

307 -

317 -

320 -

319 -

326 -

323 -

328 -

332 -

335 -

338 -

PRE-PAIRING PERIOD

Day 12 MEAN ST.DEV.

Day 13 MEAN ST.DEV.

Day 14 MEAN ST.DEV. N

PAIRING PERIOD

Day 1 MEAN ST.DEV.

Day 2 MEAN ST.DEV.

Day 3 MEAN ST.DEV.

Day 4 MEAN ST.DEV.

Day 5 MEAN ST.DEV.

Day 6 MEAN ST.DEV.

Day 7 MEAN ST.DEV.

Day 8 MEAN ST.DEV. N

338

339

344

11 10

337

339

342

344

347

351

347

350

12 10

332 -

335 -

12 10

331 -

332 -

335 -

336 -

339 -

342 -

338 -

343 -

345 -

346 -

350 -

14 10

345 -

348 -

350 -

352 -

355 -

357 -

356 -

359 -

338 -

339 -

342 -

13 10

338 -

340 -

341 -

343 -

347 -

344 -

347 -

Day 9 MEAN ST.DEV.

Day 10 MEAN ST.DEV. N

AFTER PAIRING PERIOD

Day 1 MEAN ST.DEV.

Day 2 MEAN ST.DEV.

Day 3 MEAN ST.DEV.

Day 4 MEAN ST.DEV.

Day 5 MEAN ST.DEV.

Day 6 MEAN ST.DEV. N

354

356

12 10

356

361

362

360

362

371

14 10

345 -

348 -

17 10

349 -

353 -

355 -

351 -

356 -

362 -

361 -

364 -

17 10

366 -

370 -

371 -

370 -

373 -

380 -

349 -

352 -

12 10

351 -

355 -

356 -

355 -

358 -

363 -

Table 6. Summary of Female body weight gain

Periods

Group 1 (0 mg/kg bw/day)

Group 2 (100 mg/kg bw/day)

Group 3 (300 mg/kg bw/day)

Group 4 (1000 mg/kg bw/day)

Pre-pairing period

Day 1 MEAN ST.DEV.

Day 2 MEAN ST.DEV.

Day 3 MEAN ST.DEV.

Day 4 MEAN ST.DEV.

Day 5 MEAN ST.DEV.

Day 6 MEAN ST.DEV.

Day 7 MEAN ST.DEV.

Day 8 MEAN ST.DEV.

Day 9 MEAN ST.DEV.

Day 10 MEAN ST.DEV.

Day 11 MEAN ST.DEV. N

201 8

205 7

203 7

207 8

207 9

209 8

207 8

212

214

214 8

215 8

198 -

202 -

204 -

207 -

206 -

208 -

211 -

213 -

214 -

219 -

198 -

203 -

204 -

208 -

205 -

208 -

209 -

213 -

214 -

221 -

202 -

206 -

208 -

211 -

208 -

212 -

211 -

214 -

215 -

221 -

Day 12 MEAN ST.DEV.

Day 13 MEAN ST.DEV.

Day 14 MEAN ST.DEV. N

PAIRING PERIOD

Day 1 MEAN ST.DEV.

Day 2 MEAN ST.DEV.

Day 3 MEAN ST.DEV.

Day 4 MEAN ST.DEV.

Day 5 MEAN ST.DEV.

Day 6 MEAN ST.DEV.

Day 7 MEAN ST.DEV.

Day 8 MEAN ST.DEV. N

219

221

227

228

236

238

249

251

253

258

0 1

218 -

220 -

219 -

11 10

226 -

232 -

235 -

240 -

239 -

243

245

0 1

221 -

220 -

221 -

16 10

228 -

231 -

230 -

246 -

249 -

250 -

---

221 -

219 -

220 -

12 10

228 -

226 -

---

Pairing period

Day 9 MEAN ST.DEV.

Day 10 MEAN ST.DEV. N

GESTATION PERIOD

Day 0 MEAN ST.DEV.

Day 1 MEAN ST.DEV.

Day 2 MEAN ST.DEV.

Day 3 MEAN ST.DEV.

Day 4 MEAN ST.DEV.

Day 5 MEAN ST.DEV.

Day 6 MEAN ST.DEV.

Day 7 MEAN ST.DEV.

Day 8 MEAN ST.DEV. N

263

265

0 1

227

231

235

239

244

247

249

253

258

14 8

252

251

0 1

228 -

234 -

240 -

242 -

243 -

249 -

254 -

257 -

263 -

16 8

---

228 -

234 -

240 -

244 -

248 -

252 -

256 -

258 -

264 -

---

227 -

232 -

237 -

239 -

241 -

245 -

249 -

253 -

256 -

Day 20 MEAN ST.DEV.

Day 21 MEAN ST.DEV. N

LACTATION PERIOD

Day 1 MEAN ST.DEV.

Day 2 MEAN ST.DEV.

Day 3 MEAN ST.DEV.

Day 4 MEAN ST.DEV. N

352

362

23 8

273

275

280

286

13 8

359 -

373 -

26 8

278 -

279 -

281 -

290 -

19 8

357 -

369 -

31 10

278 -

288 -

285 -

295 -

349 -

364 -

18 10

267 -

272 -

277 -

284 -

*/**/- : DUNNETT-Test based on pooled variance sig. at 5% (*), 1% (**) or not sig. (-)

Table 7. Mating performance

Days of the pairing period.	Group 1 mg/kg	Group 2 mg/kg	Group 3 mg/kg	Group 4 mg/kg
1 2 3 4 5 7 11	4 - 3 1 1 - 1	- 3 1 4 - 1 1	1 1 5 2 - 1 -	- 1 7 2 - - - -
Median precoital time	3	4	3	3
Mean precoital time	3.3	4.3	3.3	3.1
N	10	10	10	10

Table 8. Fertility parameters

Days of Parameters	Group 1 mg/kg	Group 2 mg/kg	Group 3 mg/kg	Group 4 mg/kg
Mating (%)	100	100	100	100
Fertility index (%)	80	80	100	100
Conception index (%)	80	80	100	1000
Gestation index – Breeding (%)	100	100	100	100

Percentage mating = (Females mated / Females paired) * 100
Fertility index = (Females achieving a pregnancy / Females paired) * 100
Conception rate = (Females achieving a pregnancy / Females mated) * 100
Gestation index = (Number of females with living pups / Number of females pregnant) * 100

Table 9. Summary of mean breading data per group.

Parameters	Group 1 mg/kg	Group 2 mg/kg	Group 3 mg/kg	Group 4 mg/kg
Total Litters Mean duration of gestation	8 21.4	8 21.5	10 21.7	10 21.5
Mean implantation	13.3	13.5	14.0	13.3
Post implantation loss	1.4	0.8	1.4	0.5
Dead pups at first litter check	0.3	0.3	0.0	0.0
Live pups at first check	11.9	12.8	12.6	12.8
Postnatal loss days 0- 4	0.1	0.0	0.0	0.0
Live pups day 4	11.8	12.8	12.6	12.8
Birth index (#)	89.6	94.4	90.0	96.2#
Viability index (#)	98.9	100	100	100

# / ## : Fisher's Exact Test significant at 5% (#) or 1% (##) level

Birth index = (number of pups born alive / number of implantations) * 100

Viability index = (number of alive pups on day 4 p.p. / number of pups born alive) * 100

Table 9. summary of organ/bosy weight ratio and percentage mean values.

Organs

Group 1 (0

mg/kg bw/day

Group 2 (100

mg/kg bw/day

Group 3 (300

mg/kg b/day

Group 4 (1000

mg/kg bw/day)

Body weight

366.7

14.4

357.3

20.6

373.8

18.6

360.8

13.5

Testis (L)

1.78

0.49

1.72

0.48

2.00

0.54

1.92

0.53

Testis (R)

1.75

0.48

1.67

0.47

1.92

0.52

1.92

0.53

Epididi (R)

0.681

0.186

0.642

0.180

0.730

0.195

0.714

0.198

Epidid (L)

0.675

0.185

0.644

0.180

0.743

0.199

0.738

0.205

Conclusions:

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:

In this study, an OECD 421 "“Reproduction /Developmental Toxicity Screening Test” performed to GLP was conducted to generate preliminary information concerning the effects of the test item, Sodium Sulphate, on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition.
Four groups (10 males & 10 females) were treated (by gavage) with Sodium Sulphate once daily. Group 1 was the purified water control, group 2, 3 and 4 were dosed at 100, 300 and 1000 mg/kg bw/day, respectively. All were dosed at a dose volume of 10 mL/kg bw. 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 pre-pairing, pairing, gestation and lactation period up to day 4 post partum.
In parental animals (P0), all animals survived until the scheduled necropsy and no clinical signs were observed. Mean food consumption was not affected by treatment in either sex. No test item-related effects on mean body weight and mean body weight gain were observeed in either sex. Mating performance, fertility index and conception rate were not affected by treatment. The mean number of corpora lutea, the mean number of implantations per dam, and the post-implantation losses were unaffected by test item treatment. The mean duration of gestation was unaffected by treatment. Mean weight of testes and epididymides were not affected by the treatment. All organs and tissues examined did not reveal any macroscopic or microscopic changes related to treatment.

In F1 pups, the number of live pups at first litter check and the mean litter size was unaffected by test item treatment. Sex ratios at first litter check and on day 4 post partum were unaffected by treatment. Mean pup weights and weight gains were also not considered to be affected.

As no effects were observed, the NOEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 1000 mg/kg/day.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Sulphur trioxide readily reacts with water to form sulphuric acid. The reaction is instantaneous, to the extent that SO3 will react with water vapour in the atmosphere to form fumes of sulphuric acid. This reaction forms the basis of the main manufacturing process of H2SO4. Similarly, sulphur trioxide will rapidly form sulphuric acid at the site of contact (skin, respiratory tract) due to the presence of moisture. The read-across hypothesis is therefore that SO3 will instantaneously transform into H2SO4 upon contact with water, thus any observed effects will be directly attributable to sulphuric acid. No studies are proposed for scientific reasons or reasons of animal welfare given the corrosive nature of sulphuric acid. As structurally sulphuric acid and sodium sulphate differ only by the cation this approach is considered acceptable. The data therefore adequately covers the toxicity endpoint following exposure to sodium sulphate. It is therefore justifiable to derive hazard conclusions from sulphuric acid data, with regard to toxicological endpoints.

Reason / purpose for cross-reference:

read-across source

Sex:

male/female

Vehicle:

water

Remarks:

Purified water

Analytical verification of doses or concentrations:

yes

Parental animals: Observations and examinations:

Oestrous cyclicity (parental animals):

Sperm parameters (parental animals):

Litter observations:

Postmortem examinations (parental animals):

Postmortem examinations (offspring):

Statistics:

Reproductive indices:

Offspring viability indices:

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Reproductive performance:

no effects observed

Description (incidence and severity):

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: An absence of any adverse effect, therefore the NOEL was established at 1000 mg/kg/day.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Key result

Dose descriptor:

other:

Generation:

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No findings gave any indication of a test item-related effect.

Remarks on result:

not determinable due to absence of adverse toxic effects

Reproductive effects observed:

Treatment related:

Conclusions:

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:

As no effects were observed, the NOEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 1000 mg/kg/day.

Reference 3

Endpoint:: one-generation reproductive toxicity
Type of information:: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:: weight of evidence
Justification for type of information:: No studies are proposed for scientific reasons or reasons of animal welfare given the corrosive nature of sulphuric acid. When added to water, sulphuric acid rapidly dissociates to the hydrogen and sulphate ions (pKa = 1.92), with the hydrogen ion responsible for localised irritation and corrosivity toxicity. As structurally sulphuric acid and sodium sulphate differ only by the cation this approach is considered acceptable. The data therefore adequately covers the reproductive toxicity endpoint following high exposure to sulphate in drinking water.
Reason / purpose for cross-reference:: read-across source
Specific details on test material used for the study:: Available study data for sodium sulphate is being used for read-across to the target substance, sulphur trioxide.
Clinical signs:: no effects observed
Mortality:: no mortality observed
Body weight and weight changes:: effects observed, non-treatment-related
Reproductive function: oestrous cycle:: no effects observed
Reproductive function: sperm measures:: not examined
Reproductive performance:: effects observed, non-treatment-related
: Key result
Dose descriptor:: NOAEL
Effect level:: 5 000 ppm
Based on:: test mat.
Sex:: female
Remarks on result:: not determinable because of methodological limitations
: Key result
Critical effects observed:: not specified
Clinical signs:: no effects observed
Mortality / viability:: no mortality observed
Body weight and weight changes:: effects observed, non-treatment-related
Food consumption and compound intake (if feeding study):: not examined
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: effects observed, non-treatment-related
Ophthalmological findings:: not examined
Haematological findings:: not examined
Clinical biochemistry findings:: no effects observed
Urinalysis findings:: not examined
Sexual maturation:: not examined
Anogenital distance (AGD):: not examined
Nipple retention in male pups:: not examined
Organ weight findings including organ / body weight ratios:: not examined
Gross pathological findings:: not examined
Histopathological findings:: not examined
Other effects:: not specified
Behaviour (functional findings):: not examined
Developmental immunotoxicity:: not examined
: Key result
Dose descriptor:: NOAEL
Generation:: F1
Effect level:: 5 000 ppm
Based on:: test mat.
Sex:: male/female
Remarks on result:: not determinable because of methodological limitations
: Key result
Critical effects observed:: not specified
: Key result
Reproductive effects observed:: not specified
Conclusions:: No effect on reproductive performance was observed in female mice following the ad libitum ingestion of sulfate (from sodium sulfate) in drinking water at up to 5000 ppm during gestation or lactation over two parities.
Executive summary:: In this study, six treatment groups of 10 female mice were administered water (ad libitum) as either: (0)Control (deionized water), (0)Sodium control (in deionised water) or 625 ppm, 1250 ppm, 2500 ppm or 5000 ppm sulfate. Concentrations were adapted by the addition of sodium bicarbonate content. Premating exposure of parental females to treated water was for 1 week prior to the addition of males used for mating, on confirmation of mating males were removed. All parental females were dosed throughout until study termination. Each offspring was treated from day 21 post-partum until study termination. No effect on reproductive performance were observed in female mice during gestation or lactation over two parities. Sulfate (from sodium sulfate) administered at concentrations up to 5000 ppm (ad libitum in drinking water) caused no effect on litter size, litter weaning weight, or gestational or lactational weight gain of dams over two parities. Gestational and lactational gain of dams
was not affected by level of sulfate consumed. Minor decreases in water consumption with increasing treatment (compared to the sodium control group) were noted during both parities. Also, weight gain in dams was greater during the first parity and a quadratic response to sulfates in litter weaning weights was observed in the second parity. There were no adverse effects on reproductive performance.

Reference 4

Endpoint:

one-generation reproductive toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

Published 1979

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Justification for type of information:

ANALOGUE APPROACH: Read-across from the source substance "Sulphuric acid" to the target substance to "Sulphur trioxide".
No studies are proposed for scientific reasons or reasons of animal welfare given the corrosive nature of sulphuric acid. When added to water, sulphuric acid rapidly dissociates to the hydrogen and sulphate ions (pKa = 1.92), with the hydrogen ion responsible for localised irritation and corrosivity toxicity. As structurally sulphuric acid and sodium sulphate differ only by the cation this approach is considered acceptable. The data therefore adequately covers the reproductive toxicity endpoint following high exposure to sulphate in drinking water.

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption did not differ during gestation but increased during lactation as salt level increased

Reproductive function: oestrous cycle:

not examined

Reproductive performance:

no effects observed

No significant differences in gestation or lactation gains and number
or weight of pigs at birth or at weaning were observed. Fecal consistency was normal in all treatments. Water consumption did not differ during gestation but
increased during lactation as salt level increased. Results suggest that sulfates up to 3320 ppm in water have no significant effect on reproduction in the gilt or sow.

Key result

Dose descriptor:

NOAEL

Effect level:

3 320 ppm

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable because of methodological limitations

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

After 28-days exposure to weaned pigs, no significant treatment differences in average daily gain
or feed to gain ratio were noted. Water consumption was greater among pigs that received water with added sulfates. No differences were observed in pigs that received water containing sodium sulfate or equal parts of sulfate from sodium and magnesium sulfate.

Key result

Dose descriptor:

NOAEL

Generation:

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable because of methodological limitations

Key result

Critical effects observed:

not specified

Key result

Reproductive effects observed:

not specified

Effect Of Sulfate Content Of Water On Reproductive Performance

Parameter	0(Control)	3000 ppm Na Sulfate	3000 ppm Mg & Na Sulfate
No. of pigs ^a	16	18	17
Avg initial wt. (kg)	7.5	8.0	7.7
Avg final wt. (kg)	13.4	15.0	13.8
Avg daily gain (kg)	0.21	0.25	0.22
Feed to gain ratio	2.25	2.05	2.18
Avg daily water consumption (l)	1.25 ^b	1.89 ^c	1.63 ^d
Avg fecal condition ^e	1.7 ^f	3.3 ^g	3.6 ^g

a: Three replications of six pigs per treatment. Three pigs died, data not included.
b, c, d: Means on the same line with different superscripts are significantly different (P<.05).
e: Based on a score of 1 to 5, with 1 being firm.
f, g: Means on same line with different superscripts are significantly different (P<.01).

Conclusions:

Study results suggest that sulfates up to and including 3320 ppm in water have no significant
effect on reproduction either in the gilt or sow. Furthermore, no significant treatment differences occurred in average daily gain or feed and gain ratio in offspring after weaning administered high sulfate levels of 3000 ppm with sulfate (from sodium sulfate) or 3000 ppm sulphate (equally from sodium and magnesium sulfate).

Executive summary:

In this study, thirty-one sows and 27 gilts were each assigned to three treatments groups to study the effect of water quality during gestation and lactation. Sodium sulfate was added to the water to give sulfate at 320 ppm, 1820 ppm and 3320 ppm. Water was offered ad libitum from about 30 days postbreeding through 28 days lactation. It was concluded that sulfate content of water consumed during
gestation had no significant effect on gestation gain, number of pigs per litter at birth (total and live) or average pig and litter birth weights. Sulfate in water during lactation also had no sigificant effect on lactation gain, number of pigs at 28 days and average pig and litter weights at 28 days. The ingestion of sulfates in water upto 3320 ppm had no effect of reproductive performance. When water quality was evaluated using the offspring after weaning, no significant differences occurred at 28 days in average daily gain or feed to gain ratio among weaned pigs that consumed 3000 ppm sulfate (from sodium sulfate) or 3000 ppm sulphate (equally from sodium and magnesium sulfate).

Reference 5

Endpoint:

one-generation reproductive toxicity

Type of information:

other: Journal publication

Adequacy of study:

weight of evidence

Study period:

Published 1979

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

no guideline followed

Principles of method if other than guideline:

To determine if sulfate in drinking water affects the reproductive performance of female mice during gestation and lactation over two parities. Sixty random-bred albino mice were assigned to six sulfate treatments. Sodium sulfate was added to deionized distilled water to give sulfate levels of: 0 (control), 0 (Na control); 625 ppm; 1250 ppm, 2500 ppm and 5,000. Treatments from 625 - 5000 ppm sulfate contained the same Na content (2,392 ppm) by varying the amount of Na bicarbonate content.
Parameters evaluated were water consumption, litter size, litter weaning weight, gestational and lactational weight gain of dams.

GLP compliance:

Limit test:

Justification for study design:

- Premating exposure duration for parental (P0) animals: Water was offered (ad libitum) from approximately 30 days post breeding through to lactation day 28.
- Basis for dose level selection: Three sulfate concentrations, 320 ppm, 1820 ppm and 3320 ppm
- Inclusion/exclusion of extension of Cohort 1B. No, non-standard study (1989) which does not meet current guideline requirements.
- Termination time: Not stated
- Route of administration: Ad libitum via drinking water
- Other considerations:
Low number of pigs per group which does not reflect current guidance requirements.
Limited reproductive parameters recorded which does not reflect current guidance requirements.
The pig is considered an applicable experimental model.

In addition, an evaluation of the effect of water quality on offspring after weaning was examined. 54 four week-old pigs averaging 7.5 to 8.0 kg were assigned into nine groups. Each group consisted of two pigs from each of the three sow treatments. These groups were randomly allotted to three replications of three treatments:
1: Control water
2: 3000 ppm of added sulfate from sodium sulfate
3: 3000 ppm of added sulfate supplied equally from magnesium and sodium sulfate.
Each 2.4 x 3 m pen contained six pigs. All pigs were offered water and an 18% protein, fortified corn-soybean meal diet ad libitum for the 28-day trial.

Specific details on test material used for the study:

Sulfate in the form of analytical grade sodium sulfate was added as appropriate to achieve the desired concentrations.

Species:

pig

Strain:

not specified

Details on species / strain selection:

31 sows and 27 gilts of Hampshire, Yorkshire and Duroc breeds.

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- The reproductive trial involved 31 sows and 27 gilts, grouped separately on the basis of ancestry and weight.
- Source: Not stated
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Not stated
- Weight at study initiation: Not stated
- Fasting period before study: Not stated
- Housing: Housed in unsulfated , wooden, colony type houses located in dry lots.
- Diet (ad libitum): No, each animal was restricted to 1.8 kg of feed/per day via individual feeding stalls. Fortified corn-soyabean meal diets with 10% alfalfa meals (gestation) and 10% beet pulp (lactation) included 0.5% trace mineralized salt. Crude protein content was 12.65% and 15.70% respectively.
- Water (ad libitum): yes, available from 227 litre circular tank waterers.
Self-feeders containing the lactation diet and the 227 waterers were located distant to their housing. Animals were allowed access for 2 hours in the morning and 1.5 hours in the evening.
- Acclimation period: Not stated
ENVIRONMENTAL CONDITIONS
- Temperature (°C), Humidity (%), Photoperiod (hrs dark / hrs light): Not stated
IN-LIFE DATES: From: To: Not stated

Route of administration:

oral: drinking water

Vehicle:

water

Remarks:

The local water supply was used both for the control and for making the experimental waters.

Details on exposure:

In sows and gilts water (containing sulfates at 320, 1820 and 3320 ppm) was offered (ad libitum) from approximately 30 days post breeding through to lactation day 28.

To examine the effects of high sulfate levels on offspring after weaning, 54 weaned pigs representing the above three sow treatments were given water with 0 sulfates (Control), or 3,000 ppm sulfate (from sodium sulfate ) or 3,000 ppm sulfate (from magnesium and sodium sulfate, 1:1 ratio) for a 28-day period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sulfate content was determined weekly by turbidimetric method. Averages with their standard deviations for the entire experimental period were: Control, 320 (±24) ppm; low sulfate: 1790 (±35) ppm and high sulfate, 3298 (±139) ppm.

Duration of treatment / exposure:

For 30 days post-breeding sows and gilts were randomly assigned to three groups, a control group (320 ppm sulfate), a low sulfate group (1820 ppm sulfate) and high sulfate group (3320 ppm sulfate). Water was available ad libitum from 30-days post-breeding through to 28-days post-lactation. During gestation, feed was restricted to a daily amount of 1.8 kg/per head with sows allowed access for 2 hrs (am) and 1.5 hrs (pm). After parturition, offspring at 4-weeks were placed into three treatment groups to evaluate sulfate administration to offspring. The groups were, 1: (Control water), 2: (3000 ppm sulfate from sodium sulfate) and 3: (3000 ppm sulfate, 1:1 magnesium and sodium sulfate) for an exposure period of 28-days.

Frequency of treatment:

Exposure period was daily (ad libitum in water) from 30-days post breeding to 28-days post-lactation.

Details on study schedule:

The trial involved 31 sows and 27 gilts (grouped separately) which were randomly assigned to three treatment groups ca. 30 days post breeding. Local water was used as the control and for treatment formulations. The three three treatment groups were 320 ppm (control), 1820 ppm (low sulphate) and 3320 ppm (high sulfate). Analysis used a turbidimetric method. During gestation, feed was restricted to 1.8 kg/animal daily with water available ad libitum. Sows were allowed access to feed and water each morning and evening for 2 hrs and 1.5 hrs respectively. Saline water was available to pigs from a creep area after 10 days of age.

At parturition, the number of live and stillborn, litter weight and average pig weight were recorded. Litter weight at 14 days, number of pigs at 28 days, litter weight and average pig weight at 28 days were recorded.

To determine the effect of water quality on offspring after weaning, 54 four-week-old pigs were allotted to three treatment groups: a control group (water), a sulfate group of 3000 ppm of added sulfate from sodium sulfate and a high sulfate group of 3000 ppm of added sulfate 1:1 from magnesium and sodium sulfate. All pigs were offered water and diet ad libitum for the 28-day trial.

Dose / conc.:

320 ppm (nominal)

Remarks:

Control

Dose / conc.:

1 820 ppm (nominal)

Remarks:

Sulfate (Low) treatment group

Dose / conc.:

3 320 ppm (nominal)

Remarks:

Sulfate (High) treatment group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Three sulphate groups,
1: Control: Local water - Sulfate 320ppm
2: Sulfate (Low) group - 1820 ppm
3: Sulfate (High) group - 3320 ppm
- Rationale for animal assignment (if not random): Random
- Fasting period before blood sampling for clinical biochemistry: Not stated

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes

Litter observations:

At parturition, the number of live and stillborn pigs as well as litter weight and average pig weight were obtained.
Litter weight at 14 days, number of pigs at 28 days, litter weight and average pig weight at 28 days were recorded.

Statistics:

Data were analyzed by least squares analysis of variance.

Reproductive indices:

Sulfates up to and including 3320 ppm in water have no significant effect on reproduction performance in the gilt or sow.

Offspring viability indices:

Sulfate content of water consumed during gestation had no significant effect on gestation gain, number of pigs per litter at birth (total and live) or average pig and litter birth weights.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption did not differ during gestation but increased during lactation as salt level increased

Reproductive function: oestrous cycle:

not examined

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

3 320 ppm

Based on:

test mat.

Sex:

female

Remarks on result:

not determinable because of methodological limitations

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Behaviour (functional findings):

not examined

Key result

Dose descriptor:

NOAEL

Generation:

Effect level:

3 000 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable because of methodological limitations

Key result

Critical effects observed:

not specified

Key result

Reproductive effects observed:

not specified

Effect Of Sulfate Content Of Water On Reproductive Performance

Parameter	0(Control)	3000 ppm Na Sulfate	3000 ppm Mg & Na Sulfate
No. of pigs ^a	16	18	17
Avg initial wt. (kg)	7.5	8.0	7.7
Avg final wt. (kg)	13.4	15.0	13.8
Avg daily gain (kg)	0.21	0.25	0.22
Feed to gain ratio	2.25	2.05	2.18
Avg daily water consumption (l)	1.25 ^b	1.89 ^c	1.63 ^d
Avg fecal condition ^e	1.7 ^f	3.3 ^g	3.6 ^g

a: Three replications of six pigs per treatment. Three pigs died, data not included.
b, c, d: Means on the same line with different superscripts are significantly different (P<.05).
e: Based on a score of 1 to 5, with 1 being firm.
f, g: Means on same line with different superscripts are significantly different (P<.01).

Conclusions:

Executive summary:

Reference 6

Endpoint:: one-generation reproductive toxicity
Type of information:: other: Journal publication
Adequacy of study:: weight of evidence
Study period:: Published 1989
Reliability:: 3 (not reliable)
Rationale for reliability incl. deficiencies:: significant methodological deficiencies
Remarks:: Parental male were not treated and the exposure duration for parental female was not clear. Further more the study did not follow any specific guideline was not conducted to GLP standard. Postmortem or histopathological examinations were not performed.
Qualifier:: no guideline followed
Principles of method if other than guideline:: To determine if sulfate in drinking water affects the reproductive performance of female mice during gestation and lactation over two parities. Sixty random-bred albino mice were assigned to six sulfate treatments. Sodium sulfate was added to deionized distilled water to give sulfate levels of: 0 (control), 0 (Na control); 625 ppm; 1250 ppm, 2500 ppm and 5,000. Treatments from 625 - 5000 ppm sulfate contained the same Na content (2,392 ppm) by varying the amount of Na bicarbonate content.
Parameters evaluated were water consumption, litter size, litter weaning weight, gestational and lactational weight gain of dams.
GLP compliance:: no
Limit test:: no
Justification for study design:: - Premating exposure duration for parental (P0) animals: Treated water was available ad libitum beginning 1 week prior to breeding and was continued throughout the experiment.
- Basis for dose level selection: Selected dose levels of sulfate up to 5000 ppm.
- Inclusion/exclusion of extension of Cohorts. No, non-standard study (1989) which does not meet current guideline requirements.
- Termination time for F2: Not stated
- Route of administration: Ad libitum via drinking water

- Other considerations:
Low number of mice per group which does not reflect current guidance requirements.
Limited reproductive parameters recorded which does not reflect current guidance requirements.
The mouse is considered an applicable experimental model for the sow.
Specific details on test material used for the study:: Sulfate in the form of reagent grade sodium sulfate. Sodium concentration was determined by varying the amount of sodium bicarbonate added.
Species:: mouse
Strain:: ICR
Remarks:: 60 random bred ICR virgin female mice were used.
Details on species / strain selection:: Outbred strain of albino mouse purchased from Harlan Sprague Dawley, Inc. Indianapolis, IN , USA.
Sex:: female
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc. Indianapolis (IN), USA.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Not stated
- Weight at study initiation: Not stated
- Fasting period before study: Not stated
- Housing: Housed individually in stainless steel cages with bedding
- Diet (ad libitum): yes, Commercially prepared breeders block (Wayne Mouse Breeder Blox, Wayne Pet Food Division, Continental Grain Co. Chicago ( IL), USA)
- Water (ad libitum): yes
- Acclimation period: 1 week
ENVIRONMENTAL CONDITIONS
- Temperature (°C): Environmentally controlled, ambient temperature 23.6°C
- Humidity (%): Environmentally controlled, ambient humidity 55%
- Air changes (per hr): Not stated
- Photoperiod (hrs dark / hrs light): 12-Hrs light/12-Hrs dark
IN-LIFE DATES: From: To: Not stated
Route of administration:: oral: drinking water
Vehicle:: water
Remarks:: Deionized, distilled water
Details on exposure:: Exposure period: 1 week prior to breeding until study termination
Details on mating procedure:: After 1 weeks acclimation to the treatments, a male mouse that had received tap water was paired randomly with each female mouse. The females were checked every 24 h in the morning for the presence of a vaginal plug. If one was observed, the male mouse was removed and the female weighed.
Analytical verification of doses or concentrations:: no
Duration of treatment / exposure:: 1). Premating exposure of parental females to treated water was for 1 week prior to breeding.
2). There was no premating exposure period for males used for mating, on confirmation of mating the respective male removed from the cage.
3). All parental females dosed throughout until study termination
4): Each offspring was treated from day 21 post-partum until study termination
Frequency of treatment:: Daily, drinking water containing sulfate was available ad libitum
Details on study schedule:: After 1 week of treatment acclimation to parental females a male mouse having received tap water was paired randomly with each female mouse. The females were checked every 24h (morning) for the presence of a vaginal plug. If observed, the male mouse was removed and the female weighed. Water consumption was measured daily, during the 2nd and 3rd week of gestation and the 1st and 2nd week of lactation.

Measurements of water consumption during week 1 of gestation were omitted due to excessive drinking bottle leakage. Water consumptions during week 3 of lactation were omitted because the pups could begin drinking water and interfere with accurate measurement of female
water consumption. At parturition, dams were weighed and litter size recorded. The litters then were standardized to eight pups per litter. At day 21 postpartum, the pups were weaned and the litters and dams were weighed individually. The dams then were rebred at first estrus immediately following weaning. This procedure was carried out over two parities. Only animals that whelped during each parity were used in the analysis.
Dose / conc.:: 0 ppm (nominal)
Remarks:: Control (deionized, distilled water)
Dose / conc.:: 0 ppm (nominal)
Remarks:: Sodium control (in deionised, distilled water)
Dose / conc.:: 625 ppm (nominal)
Remarks:: Sulphate (Low) treatment group
Dose / conc.:: 1 250 ppm (nominal)
Remarks:: Sulfate treatment group
Dose / conc.:: 2 500 ppm (nominal)
Remarks:: Sulfate treatment group
Dose / conc.:: 5 000 ppm (nominal)
Remarks:: Sulfate (High) treatment group
No. of animals per sex per dose:: First parity:
Control : 6 dams
Sodium control: 6 dams
625 ppm: 4 dams (Low dose)
1250 ppm: 5 dams
2500 ppm: 9 dams
5000 ppm: 7 dams (High dose)

Second parity:
Control : 4 dams
Sodium control: 4 dams
625 ppm: 4 dams (Low dose)
1250 ppm: 4 dams
2500 ppm: 4 dams
5000 ppm: 4 dams (High dose)
Control animals:: yes, concurrent no treatment; yes, concurrent vehicle
Details on study design:: - Dose selection rationale: Six sulphate groups (treatment groups 2-6 contained the same Na content (i.e. 2392 ppm, by varying the amount of added sodium bicarbonate)
1: Control: Deionised water - 0 ppm
2: Sodium control: Deionised water - 0 ppm
3: Sulfate - 625 ppm
4: Sulfate - 1250 ppm
5: Sulfate - 2500 ppm
6: Sulfate - 5000 ppm

- Rationale for animal assignment: Random
- Fasting period before blood sampling for clinical biochemistry: Not stated
Parental animals: Observations and examinations:: CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
At parturition, dams were weighed and litter size recorded.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Water consumption was measured daily (graduated cylinder), during the 2nd and 3rd week of gestation and the 1st and 2nd week of lactation. Measurements of water consumption during week 1 of gestation were omitted due to excessive water leakage. Water consumptions during week 3 of lactation were omitted because the pups began drinking water and thus interfere with accurate measurement of female water consumption.
Oestrous cyclicity (parental animals):: Dams were rebred at first estrus immediately following weaning.
Litter observations:: At parturition, dams were weighed and litter size was recorded. The litters then were standardized to eight pups/litter. Dams that whelped fewer than eight pups received pups from dams with greater than eight pups within the same treatment. When this was not possible, pups were cross-fostered pups from dams that were on a lower sulfate treatment. At 21 d post-partum, the pups
were weaned and the litters and dams were weighed individually. The dams then were rebred at first estrus immediately following weaning. This procedure was carried out over two parities. Only animals that whelped during each parity were used in the analysis.

During both parities, the litter size of live pups equalled the total number born as no mortalities at birth were detected.

During both parities, there were no differences (P > .10) in the litter size among treatments. The 21-d litter weaning weight for both parities were not affected (P > .10) by the sulfate treatment. A
quadratic response (P < .005) in weaning weight was observed within sulfate treatments during the second parity.
Postmortem examinations (parental animals):: SACRIFICED: Not stated
GROSS NECROPSY: Not performed
HISTOPATHOLOGY / ORGAN WEIGHTS: Not performed
Postmortem examinations (offspring):: SACRIFICED: Not stated
GROSS NECROPSY: Not performed
HISTOPATHOLOGY / ORGAN WEIGHTS: Not performed
Statistics:: The least squares mean analysis of variance technique (SAS, 1982) was used to analyze the
data. One contrast was used to compare the deionized, distilled water control treatment to
all sulfate treatments. Sulfate treatment (2 to 6) effects were partitioned into linear, quadratic
and cubic components using orthogonal contrasts. Student's t-test (Steel and Torrie, 1980)
were used to determine the difference in water consumption between weeks in both gestation
and lactation.

Cited statistical references:
1: SAS (1982). SAS User's Guide: Statistics. SAS Inst., Inc., Cary, NC.

2: Steel, R.G.D. and J. H. Torrie. (1980). Principles and Procedures of Statistics. McGraw Hill Book Co., St. Louis, MO.
Reproductive indices:: No effect on reproductive performance when litter size and weaning weight are used as indicators of maximum reproductive performance. Sulfates in the water up to 5000 ppm (ad libitum) do not affect reproductive performance in the gestating mouse.
Offspring viability indices:: During both parities, the litter size of live pups equalled the total number born as no mortalities at birth were detected.
Clinical signs:: no effects observed
Mortality:: no mortality observed
Body weight and weight changes:: effects observed, non-treatment-related
Reproductive function: oestrous cycle:: no effects observed
Reproductive function: sperm measures:: not examined
Reproductive performance:: effects observed, non-treatment-related
: Key result
Dose descriptor:: NOAEL
Effect level:: 5 000 ppm
Based on:: test mat.
Sex:: female
Remarks on result:: not determinable because of methodological limitations
: Key result
Critical effects observed:: not specified
Clinical signs:: no effects observed
Mortality / viability:: no mortality observed
Body weight and weight changes:: effects observed, non-treatment-related
Food consumption and compound intake (if feeding study):: not examined
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: effects observed, non-treatment-related
Ophthalmological findings:: not examined
Haematological findings:: not examined
Clinical biochemistry findings:: no effects observed
Urinalysis findings:: not examined
Sexual maturation:: not examined
Anogenital distance (AGD):: not examined
Nipple retention in male pups:: not examined
Organ weight findings including organ / body weight ratios:: not examined
Gross pathological findings:: not examined
Histopathological findings:: not examined
Other effects:: not specified
Behaviour (functional findings):: not examined
Developmental immunotoxicity:: not examined
: Key result
Dose descriptor:: NOAEL
Generation:: F1
Effect level:: 5 000 ppm
Based on:: test mat.
Sex:: male/female
Remarks on result:: not determinable because of methodological limitations
: Key result
Critical effects observed:: not specified
: Key result
Reproductive effects observed:: not specified
Conclusions:: No effect on reproductive performance was observed in female mice following the ad libitum ingestion of sulfate (from sodium sulfate) in drinking water at up to 5000 ppm during gestation or lactation over two parities.
Executive summary:: In this study, six treatment groups of 10 female mice were administered water (ad libitum) as either: (0)Control (deionized water), (0)Sodium control (in deionised water) or 625 ppm, 1250 ppm, 2500 ppm or 5000 ppm sulfate. Concentrations were adapted by the addition of sodium bicarbonate content. Premating exposure of parental females to treated water was for 1 week prior to the addition of males used for mating, on confirmation of mating males were removed. All parental females were dosed throughout until study termination. Each offspring was treated from day 21 post-partum until study termination. No effect on reproductive performance were observed in female mice during gestation or lactation over two parities. Sulfate (from sodium sulfate) administered at concentrations up to 5000 ppm (ad libitum in drinking water) caused no effect on litter size, litter weaning weight, or gestational or lactational weight gain of dams over two parities. Gestational and lactational gain of dams
was not affected by level of sulfate consumed. Minor decreases in water consumption with increasing treatment (compared to the sodium control group) were noted during both parities. Also, weight gain in dams was greater during the first parity and a quadratic response to sulfates in litter weaning weights was observed in the second parity. There were no adverse effects on reproductive performance.

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 relevant studies investigating the reproductive effects of sulphur trioxide or sulphuric acid exposure in animals are available. The testing of sulphur trioxide for effects on fertility in a conventional two-generation study in the rat is not proposed.

The likely corrosive nature of the substance means that testing cannot be justified for reasons of animal welfare. The usual route of exposure in the two-generation reproductive toxicity study is not relevant to human exposure as effects will be dominated by local irritation of the gastrointestinal tract. Similarly, testing of sulphur trioxide using dermal exposure cannot be justified.

In terms of worker exposure, the inhalation route is most relevant. However numerous inhalation toxicity studies performed with sulphuric acid in a number of different species and of various durations have not demonstrated any systemic effects of exposure at levels up to those causing mortality. The effects of inhalation of sulphuric acid are limited to the respiratory tract (structure and function); effects on other organs and tissues have not been identified. Specifically, no gross effects on the reproductive organs were observed in two species (rat and guinea pig) following chronic exposure. Following a comprehensive literature review, the OECD SIDS for sulphuric acid (2001) concluded that sulphuric acid '...is not expected to be absorbed or distributed throughout the body. Therefore it is not likely that it will reach male and female reproductive organs following exposures by any route'. The ATSDR (1998) comes to a similar conclusion, stating that 'Because sulfuric acid is a direct-acting toxicant and not likely to reach reproductive organs or the fetus, reproductive and developmental effects are unlikely to occur in humans exposed to sulfuric acid'.

Sulphur trioxide will rapidly react with atmospheric moisture or moisture present at the initial site of contact (skin, respiratory tract) to produce sulphuric acid. The toxicity of sulphur trioxide is therefore essentially the toxicity of sulphuric acid. As previously discussed, sulphuric acid will immediately dissociate on contact with the body to form sulphate and hydrogen (carbonium) ions. The hydrogen ion is likely to be entirely responsible for the site of contact effects of sulphuric acid. Any hydrogen ion absorbed into the systemic circulation will be subject to the normal homeostatic mechanisms governing physiological pH, and will be excreted in the urine when in excess. Sulphate ions are also a normal product of metabolism and are present in significant amounts in the body. Studies have demonstrated the absorption of sulphate following inhalation exposure to sulphuric acid, however levels of sulphate are under homeostatic control and any 'excess' sulphate will be excreted in the urine.

It is therefore considered that the testing of sulphur trioxide (or sulphuric acid) for effects on fertility cannot be justified, both on scientific grounds (due to the lack of systemic exposure) and for reasons of animal welfare (due to the corrosive nature of these substances). A waiver is therefore proposed for this endpoint.

Short description of key information:

No studies of the effects of sulphur trioxide or sulphuric acid exposure on fertility have been identified.

Effects on developmental toxicity

Description of key information

No studies of developmental toxicity have been performed with sulphur trioxide. A study of developmental toxicity in the rabbit and mouse (using inhalation exposure) has been performed with sulphuric acid, to a protocol broadly comparable to OECD 414. No evidence of teratogenicity was seen in either species, at exposure levels sufficient to cause mild maternal toxicity.

Link to relevant study records

Referenceopen all close all

Reference 1

Endpoint:: developmental toxicity
Type of information:: experimental study
Adequacy of study:: key study
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: other: Literature study, comparable to OECD 414
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:: yes
Remarks:: Number of exposure levels
Principles of method if other than guideline:: Sulphuric acid was tested by inhalation for embryotoxicity in mice according to a protocol similar to OECD guideline 414
GLP compliance:: no
Remarks:: Older, published study
Limit test:: no
Species:: mouse
Route of administration:: inhalation: aerosol
Type of inhalation exposure (if applicable):: whole body
Vehicle:: water
Analytical verification of doses or concentrations:: yes
Details on mating procedure:: Nulliparous female mice were pen-bred with fertile males of the same strain. The day on which a vaginal plug was observed in mice was considered day zero of gestation.
Duration of treatment / exposure:: 7 hours/day: Day 6-15 of gestation
Frequency of treatment:: Daily
Duration of test:: Day 18 of gestation
No. of animals per sex per dose:: 35 (with controls)
Control animals:: yes
Dose descriptor:: LOAEC
Effect level:: 19.3 mg/m³ air (analytical)
Basis for effect level:: other: maternal toxicity
Dose descriptor:: NOAEC
Effect level:: 5.7 mg/m³ air (analytical)
Basis for effect level:: other: maternal toxicity
Dose descriptor:: NOAEC
Effect level:: 19.3 mg/m³ air (analytical)
Basis for effect level:: other: developmental toxicity
Dose descriptor:: NOAEC
Effect level:: 19.3 mg/m³ air (analytical)
Basis for effect level:: other: teratogenicity
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.
Executive summary:: Mated female mice were exposed (whole body) to atmospheres containing aerosols of sulphuric acid at 0, 5.7 mg/m3 (MMAD 1.6 um) or 19.3 mg/m3 (MMAD 2.4 um) on Days 6 -15 of gestation (mice). Animals were sacrificed at Days 18 (mice). Evidence of mild toxicity was seen in maternal mice (reduced food consumption) at 20 mg/m3. No evidence of teratogenicity or developmental toxicity was seen.

Reference 2

Endpoint:: developmental toxicity
Type of information:: experimental study
Adequacy of study:: key study
Reliability:: 2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:: other: Literature study, comparable to OECD 414
Qualifier:: equivalent or similar to guideline
Guideline:: OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:: yes
Remarks:: Number of exposure levels
Principles of method if other than guideline:: Sulphuric acid was tested by inhalation for embryotoxicity in rabbits according to a protocol similar to OECD guideline 414
GLP compliance:: no
Remarks:: Older, published study
Limit test:: no
Species:: rabbit
Strain:: other: New Zealand White, CF-1
Route of administration:: inhalation: aerosol
Type of inhalation exposure (if applicable):: whole body
Vehicle:: water
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:: 7 hours/day: Day 6-18 of gestation
Frequency of treatment:: Daily
Duration of test:: Day 29 of gestation
No. of animals per sex per dose:: 20 (with controls)
Control animals:: yes
Dose descriptor:: LOAEC
Effect level:: 19.3 mg/m³ air (analytical)
Basis for effect level:: other: Maternal toxicity
Dose descriptor:: NOAEC
Effect level:: 5.7 mg/m³ air (analytical)
Basis for effect level:: other: Maternal toxicity
Dose descriptor:: NOAEC
Effect level:: 19.3 mg/m³ air (analytical)
Basis for effect level:: other: Developmental toxicity
Dose descriptor:: NOAEC
Effect level:: 19.3 mg/m³ air (analytical)
Basis for effect level:: other: Teratogenicity
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.
Executive summary:: Inseminated female rabbits were exposed (whole body) to atmospheres containing aerosols of sulphuric acid at 0, 5.7 mg/m3 (MMAD 1.6 um) or 19.3 mg/m3 (MMAD 2.4 um) on Days 6 -18 of gestation (rabbits). Animals were sacrificed at Day 29 of gestation (rabbits). Evidence of mild toxicity was seen in maternal rabbits (reduced weight gain, respiratory tract effects) at 20 mg/m3. No evidence of teratogenicity or developmental toxicity was seen.

Effect on developmental toxicity: via oral route

Endpoint conclusion:: no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEC
: 19.3 mg/m³
Study duration:: subacute
Experimental exposure time per week (hours/week):: 49
Species:: other: rabbit and mouse
Quality of whole database:: The published study, comparable to OECD 414, is considered reliable and adequate for characterising the developmental toxicity of the substance

Effect on developmental toxicity: via dermal route

Endpoint conclusion:: no study available

Additional information

No studies of the developmental toxicity of sulphur trioxide have been performed. Sulphur trioxide will rapidly react with atmospheric moisture or moisture present at the initial site of contact (skin, respiratory tract) to produce sulphuric acid. The toxicity of sulphur trioxide is therefore essentially the toxicity of sulphuric acid.

In the study by Murray, F..J. et al (1979), mated female mice were exposed (whole body) to atmospheres containing aerosols of sulphuric acid at 0, 5.7 mg/m3 (MMAD 1.6 um) or 19.3 mg/m3 (MMAD 2.4 um) on Days 6 -15 of gestation (mice). Animals were sacrificed at Days 18 (mice). Evidence of mild toxicity was seen in maternal mice (reduced food consumption) at 20 mg/m3. No evidence of teratogenicity or developmental toxicity was seen. Inseminated female rabbits were exposed (whole body) to atmospheres containing aerosols of sulphuric acid at 0, 5.7 mg/m3 (MMAD 1.6 um) or 19.3 mg/m3 (MMAD 2.4 um) on Days 6 -18 of gestation (rabbits). Animals were sacrificed at Day 29 of gestation(rabbits). Evidence of mild toxicity was seen in maternal rabbits (reduced weight gain, respiratory tract effects) at 20 mg/m3. No evidence of teratogenicity or developmental toxicity was seen.

It is calculated that exposure to sulphuric acid at the TWA will result in negligible additional systemic sulphate exposure, which will not have any effect on foetal development.

Testing cannot be justified on scientific grounds (given the absence of systemic exposure, detailed above) or (due to the corrosive nature of the substance) for reasons of animal welfare. Both the OECD SIDS (2001) and the US ATSDR (1998) concluded that the exiting information do not show any evidence of developmental toxicity and that further testing is not required based on a lack of systemic exposure.

Caccatelli, R. 2010: In parental animals (P0), all animals survived until the scheduled necropsy and no clinical signs were observed after administration of Sodium Sulphate up to 1000 mg/kg bw/day. Mean food consumption was not affected by treatment in either sex. No test item-related effects on mean body weight and mean body weight gain were observeed in either sex. Mating performance, fertility index and conception rate were not affected by treatment. The mean number of corpora lutea, the mean number of implantations per dam, and the post-implantation losses were unaffected by test item treatment. The mean duration of gestation was unaffected by treatment. Mean weight of testes and epididymides were not affected by the treatment. All organs and tissues examined did not reveal any macroscopic or microscopic changes related to treatment. In F1 pups, the number of live pups at first litter check and the mean litter size was unaffected by test item treatment. Sex ratios at first litter check and on day 4 post partum were unaffected by treatment. Mean pup weights and weight gains were also not considered to be affected. As no effects were observed, the NOEL (No Observed Effect Level) for reproduction/developmental toxicity was considered to be 1000 mg/kg/day.

Toxicity to reproduction: other studies

Additional information

No studies have ben performed and none are proposed in the absence of any effects in the existing dataset and given the absence of systemic exposure. Further studies cannot be justified on scientific grounds and (given the corrosive nature of the substance) also cannot be justified for reasons of animal welfare.

Justification for classification or non-classification

No classification is proposed for reproductive or developmental toxicity. The existing data and the absence of systemic exposure do not indicate that classification is required.

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Sulfate(ppm)	First parity^b		Second parity^b
Sulfate(ppm)	Pups/ litter	Weaning wt.(g)^c	Pups/ litter	Weaning wt.(g)^c
0 (Control) Deionized, distilled water	8.0	123.3	11.3	129.7
Na control ("0" sulfate)	8.3	128.1	13.0	130.6
625	8.8	114.3	13.3	126.9
1250	7.8	130.0	13.5	141.2
2500	8.0	132.7	13.0	144.2
5000	8.5	125.9	15.5	130.4
SE	0.6	6.2	1.3	3.8^d

Sulfate(ppm)	First parity^b		Second parity^b
Sulfate(ppm)	Pups/ litter	Weaning wt.(g)^c	Pups/ litter	Weaning wt.(g)^c
0 (Control) Deionized, distilled water	8.0	123.3	11.3	129.7
Na control ("0" sulfate)	8.3	128.1	13.0	130.6
625	8.8	114.3	13.3	126.9
1250	7.8	130.0	13.5	141.2
2500	8.0	132.7	13.0	144.2
5000	8.5	125.9	15.5	130.4
SE	0.6	6.2	1.3	3.8^d

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Sulphur trioxide

Endpoint summary

Administrative data

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Effects on fertility

Description of key information

Link to relevant study records

Referenceopen allclose all

Reference 1

Reference 2

Reference 3

Effect Of Sulfate Treatment On Litter Sizea At Weaning And Litter Weaning Weight (Both Parities)

Sulfate(ppm)

First parityb

Second parityb

Pups/litter

Weaning wt.(g)c

Pups/litter

Weaning wt.(g)c

0 (Control) Deionized, distilled water

8.0

123.3

11.3

129.7

Na control ("0" sulfate)

8.3

128.1

13.0

130.6

625

8.8

114.3

13.3

126.9

1250

7.8

130.0

13.5

141.2

2500

8.0

132.7

13.0

144.2

5000

8.5

125.9

15.5

130.4

SE

0.6

6.2

1.3

3.8d

Reference 4

Effect Of Sulfate Content Of Water On Reproductive Performance

Parameter

0(Control)

3000 ppmNa Sulfate

3000 ppmMg & Na Sulfate

No. of pigs a

16

18

17

Avg initial wt. (kg)

7.5

8.0

7.7

Avg final wt. (kg)

13.4

15.0

13.8

Avg daily gain (kg)

0.21

0.25

0.22

Referenceopen all close all

Effect Of Sulfate Treatment On Litter Size^a At Weaning And Litter Weaning Weight (Both Parities)

First parity^b

Second parity^b

Pups/
litter

Weaning wt.(g)^c

Pups/
litter

Weaning wt.(g)^c

3.8^d

3000 ppm
Na Sulfate

3000 ppm
Mg & Na Sulfate

No. of pigs ^a

1.25 ^b

1.89 ^c

1.63 ^d

Avg fecal condition ^e

1.7 ^f

3.3 ^g

3.6 ^g

3000 ppm
Na Sulfate

3000 ppm
Mg & Na Sulfate

No. of pigs ^a

1.25 ^b

1.89 ^c

1.63 ^d

Avg fecal condition ^e

1.7 ^f

3.3 ^g

3.6 ^g

Effect Of Sulfate Treatment On Litter Size^a At Weaning And Litter Weaning Weight (Both Parities)

First parity^b

Second parity^b

Pups/
litter

Weaning wt.(g)^c

Pups/
litter

Weaning wt.(g)^c