Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant OECD414 study without deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Sulphamidic acid
EC Number:
226-218-8
EC Name:
Sulphamidic acid
Cas Number:
5329-14-6
Molecular formula:
H3NO3S
IUPAC Name:
sulfamic acid
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Sulphamic Acid
- Molecular formula: H3NO3S
- Substance type: Inorganic
- Physical state: Solid
- Analytical purity: 99.9%
- Lot/batch No.: 13122201
- Expiration date of the lot/batch: December 21, 2014
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature (+10 °C to +25 °C), dry in a tightly closed container.

Test animals

Species:
rat
Strain:
other: Rat / CD / Crl:CD (SD)
Details on test animals or test system and environmental conditions:
Mature female CD® rats were used for this experiment, a strain bred by Charles River Laboratories, Research Models and Services, Germany GmbH.
Each animal was given a thorough examination. Animals were judged to be healthy on the basis of general observations (physical examination). Health checks were performed on the day of delivery and at the beginning of matings. The spare animals were retained to replace any rat showing signs of illness. No replacements had to be carried out.
Body weight (on day 0 of gestation): 196.2 - 240.8 g
Age (on day 0 of gestation): 60 days
For identification, each rat received a continuous number between 1 and 100: a pattern of points was set on paws and/or tail by tattoo. Additionally, the animal cages were labelled with the tattooed serial number, sex, study code number, type of study, route of administration, dose level, date of conception and dates of administration.
Diet: Commercial ssniff® R/Z V1324 (ssniff Spezialdiäten GmbH, 59494 Soest, Ger-many, see Appendix 2: Composition of the Diet) served as food. This food was offered daily ad libitum. Samples of the food are analysed for contaminants based on EPA/USA by LUFA-ITL at least twice a year. Certificates of analysis of the composition and for contaminants were provided by the manufacturer and are included in the raw data. No contaminants above the limitations were noted.
Housing: Except during the mating period, the dams were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. The room temperature was 22 °C ±3 °C (maximum range) and the relative humidity 55% ±15% (maximum range). No values outside the maximum range were noted during the course of the study. Granulated textured wood released for animal bedding (Granulat A2, J. Brandenburg, 49424 Goldenstedt/Arkeburg, Germany) was used as bedding material in the cages. The cages were cleaned and changed once a week. Periodic analysis of the bedding material for contaminants based on EPA/USA is conducted at least once a ear by LUFA-ITL.
The rooms were alternately lit (about 150 lux at approximately 1.5 m room height) and darkened in a 12 hours dark/12 hours light cycle.
Drinking water: Drinking water was offered ad libitum. Samples of drinking water are taken periodically by the Wasserwerk Wankendorf and periodic analyses are performed by LUFA-ITL according to the 'Deutsche Trinkwasserverordnung 2001' [German Regulations on drinking water 2001].
In addition, drinking water samples taken at LPT are analysed by LUFA-ITL once a year for means of bacteriological investigations according to the 'Deutsche Trinkwasserverordnung 2001' [German Regulations on Drinking Water 2001].

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Route of administration: Oral by gavage
Frequency of administration: Once daily
Treatment period: Day 6 to 19 of gestation
Vehicle: Water for injection
Administration volume: 10 mL/kg b.w./day
Selection of route of administration: According to OECD guideline 414
The test item formulations were freshly prepared every day. The test item was dissolved in the vehicle to the appropriate concentration and was administered orally at a constant volume once daily. The amount of the test item was daily adjusted to the current body weight of the animal. The control animals received the vehicle at the same administration volume dailly in the same way.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the test item formulations, samples of approximately 5 mL were taken at the following times and stored at -20 °C or colder until analysis:
At study initiation: Analysis of stability and concentration was done immediately after preparation of the formulation as well as after 8 and 24 hours storage of the test item preparations at room temperature (3 samples /dose level group; groups 2 - 4).
At study termination (at a time when the majority of animals was dosed) during treatment with the test item always before administration to the last animal of the group (1 sample/group).
The samples were labelled with the study number, species, type of sample, concentration, test day, sampling time and date.
The concentration of Sulphamic Acid in the test item formulation samples was determined by measuring the amount of a NaOH solution (0.01M, 0.05M or 0.15M) which was necessary to cause the colour change of the pH indicator (Phenolpthalein) in one mL test item formulation.
The following calculation was used: One mL 0.01 M NaOH is necessary to cause the colour change of the pH indicator in a solution of 1 mL test item formulation containing 0.971 mg Sulphamic Acid. Five titrations were performed from each sample of test item formulation.
The measured actual concentrations of the test item in the test item vehicle mixtures were between 102.0% and 105.7% of the nominal concentrations, indicating correctly prepared formulations which were stable at room temperature for at least 24h.
Details on mating procedure:
Four groups of pregnant rats were formed from matings, which were carried out on a daily basis. Vaginal lavages were taken each morning. When positive, the animals were assigned to the test groups by mating day using a Provantis (Version 8.2.0.8) generated randomization.
Sexually mature ('proved') male rats of the same breed served as partners. The female breeding partners were randomly chosen.
Mating was monogamous: 1 male and 1 female animal were placed together in one cage during the dark period. Each morning a vaginal smear was taken to check for the presence of sperm. If findings were negative, mating was repeated with the same partner. The day on which sperm was found was considered as the day of conception (day 0 of pregnancy). This procedure was repeated until enough pregnant dams were available for all groups. Rats which did not become pregnant (3 control animals out of 25 control animals) were excluded from the analysis of the results and replaced by spare animals. The number of non-pregnant animals of this study was within the normal range of variation. A post-mortem negative staining according to SALEWSKI was carried out in the replaced animals in order to confirm the non-pregnancy status.
Duration of treatment / exposure:
Pregnant rats were treated with the test item starting from the 6th and lasting until the 19th day of pregnancy (the 'critical' phase of organogenesis and fetal development).
Frequency of treatment:
Once daily
Duration of test:
Start of study
First mating results: February 25, 2014
First administration: March 03, 2014
Study termination
End of the in-life part: March 20, 2014
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
60 mg/kg b.w./day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
200 mg/kg b.w./day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
600 mg/kg b.w./day
Basis:
nominal in water
No. of animals per sex per dose:
25 animals per dose, 20 of them to be evaluated
Control animals:
yes, concurrent vehicle
Details on study design:
Four groups of pregnant rats were formed from matings, which were carried out on a daily basis. Vaginal lavages were taken each morning. When positive, the animals were assigned to the test groups by mating day using a Provantis (Version 8.2.0.8) generated randomization.
Justification for dose selection: The dose levels were selected in agreement with the Sponsor based on available toxicity data and a dose-range finding Study (no. 30799) performed at LPT. In the dose-range finding study, Sulphamic Acid was administered to female rats at dose levels of 100, 300 or 1000 mg/kg b.w./day orally, by gavage, once daily for 14 days. Oral treatment with 100 mg Sulphamic Acid/kg b.w./day for 14 days did not cause any signs of systemic intolerance reactions. At 300 mg Sulphamic Acid/kg b.w./day, breathing sounds were recorded for one of three intermediate dose females on two test days. Treatment with 1000 mg Sulphamic acid/kg b.w./day caused slightly or moderately reduced motility, breathing sounds or laboured breathing, increased salivation and/or a haemorrhagic nose/snout. In addition, transient reductions were noted for body weight, body weight gain and food intake. One high dose female was found dead on test day 3. Necropsy after premature death revealed gastro-intestinal lesions (multiple haemorrhagic foci at the fundus region of the stomach, inflated intestines) and hepatic changes (liver partly pale). No test item-related findings were noted for the consistency of faeces in the three females each treated with 100 or 300 mg Sulphamic acid/kg b.w./day or in the two surviving females treated with 1000 mg Sulphamic acid/kg b.w./day. The visual appraisal of the drinking water consumption revealed no test item-related influence. Macroscopic inspection during necropsy revealed no test item-related gross pathology changes. Normal values compared to the control were noted for relative and absolute organ weights of liver, kidneys and ovaries.

Examinations

Maternal examinations:
Clinical signs: Individual animals were observed daily for behaviour, external appearance and nature of the faeces. Immediately after administration, any signs of illness or reaction to treatment were recorded. In case of changes, the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m. On Saturdays and Sundays, the animals were checked regularly starting from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m. Dated and signed records of appearance, change and disappearance of clinical signs were maintained on clinical history sheets for individual animals.
Viability: Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. This would have allowed post mortem examinations to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed except that the final check was carried out at approximately midday. Animals showing signs of abortion or premature delivery would have been sacrificed on the same day. Fetuses obtained this way were examined for abnormal development, whenever possible. No abortion occurred in the study.
Body weight: The weight of each rat was recorded on day 0 of gestation (the day of detection of a positive mating sign), followed by daily weighings - always at the same time of the day. The body weight gain was also calculated in intervals (i.e. day 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-20). Furthermore the net weight change from day 6 is given. These values are stated in the report. These measurements were also used for calculating the daily amount of test item to be administered.
Food and drinking water consumption: The quantity of food consumed by each rat was recorded daily. Food intake per rat (g/rat/day) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment day. The relative food consumption (g/kg b.w./day) was calculated using the following formula: Daily food consumption [g/kg b.w./day] = Total food intake in g x 1000/Body weight in g
Daily monitoring by visual appraisal of the drinking water bottles was maintained throughout the study.
Ovaries and uterine content:
Necropsy: On the 20th day of gestation, the rats were laparotomised under ether narcosis. The ovaries and the uteri of the dams were removed; the uteri (in toto) were weighed. In order to check for possible drug effects, a dissection with macroscopic examination of the internal organs and placentae of the dams was carried out on the day of scheduled laparotomy or on the day on which the animals were found dead. In case of macroscopical findings, the affected maternal tissues were preserved in 7% buffered formalin for possible future histopathological examinations.
The fetuses were removed and the following examinations performed:
(a) Macroscopic inspection (gross evaluation) of the placentae for example for focal indurations.
(b) The number of fetuses (alive and dead) and placentae was determined
(c) Sex and viability of fetuses were determined. Animals are said to be viable when they are found alive (spontaneous breathing, spontaneous movement).
(d) Number and size of resorptions were determined.
(e) Corpora lutea in the ovaries, implantations and location of fetuses in the uterus were determined.
(g) Weights of fetuses and weights of the placentae were determined (fetuses were considered as runts if their weight was less than 70% of the mean litter weight).
(h) Fetuses were inspected externally for damages, especially for malformations .
(i) The fetuses were sacrificed by an ether atmosphere.
(j) Examination of fetuses and determination of number and kind of retardations, variations or malformations: 50% of the number of fetuses in each litter were examined for skeletal anomalies. The thorax and peritoneal cavity (without damage to ribs and sternum) were opened and the location, size and condition of the internal organs were determined. Then the skeleton was double-stained with Alcian blue for the examination of cartilage and with Alizarin red to reveal ossifications (according to DAWSON). The skeletal system was examined (determination of the number and type of retardations, variations as well as malformations). The remaining 50% of the number of fetuses in each litter were examined for soft tissue anomalies. Body sections were made and examined according to WILSON. The fetuses were allocated to the evaluation of DAWSON or WILSON on an alternating basis.
Fetal examinations:
Evaluation / parameters
Corpora lutea
- number per dam
- absolute number per group
- mean per group
Implantations
- number per dam
- distributions in the uterine horns
- absolute number per group
- mean per group
Resorptions
- number per dam, % per litter
- distributions in the uterine horns
- absolute number per group
- mean per group
- mean % per group
- early resorptions <2 mm, number and % per litter
- Late resorptions >2 mm, number and % per litter
- % litters with resorptions per group.
Weight of placentae
- individual data per fetus
- mean per litter
- mean per group
- litter mean per group
- litter mean per sex and group
Weight of fetuses
- individual data per fetus
- mean per litter
- mean per group
- litter mean per group
- litter mean per sex and group
Fetuses
- number and % per dam (alive)
- number and % per dam (dead)
- number of fetuses per sex and dam
- sex ratio per litter
- distribution in the uterine horns
- absolute number of fetuses alive per group
- mean number of fetuses alive per group
- mean % of fetuses alive per group
- mean % per sex and group
Runts
- number per dam
- mean per group
Malformed fetuses
- individual data per fetus
- type of malformation: fetal and litter incidence
- number of affected fetuses per group [%] (fetuses affected by several changes counted as one fetal incidence)
Fetuses with variations
- individual data per fetus
- type of variation: number and incidence (%) per group and litter
- Number of affected fetuses per group [%] (fetuses affected by several changes counted as one fetal incidence)
Fetuses with retardations
- individual data per fetus
- type of variation: number and incidence (%) per group and litter
- number of affected fetuses per group [%] (fetuses affected by several changes counted as one fetal incidence)
Pre-implantation loss (mean % per group; values per group)
Post-implantation loss (mean % per group; values per group)
Statistics:
The following data were captured or calculated by the the departmental computerized system (Provantis integrated preclinical software, version 8.2.0.8, Instem LSS Ltd): Clinical signs, body weight, body weight gain, food consumption, gravid uterus weight, carcass weight and the net and absolute weight change from day 6 until laparotomy. Raw data not fully compatible with the computerized sytem (e.g. reproduction data, skeletal and soft tissue examination) were maintained on paper according to the appropriate SOPs.
Statistical analyses of the parametrical values, captured or calculated by Provantis, were done by Provantis using the following settings: Analysis of normal distribution and homogeneity of variances was perfromed by using the SHAPIRO-WILKS test and the BARTLETT test. Data not normally distributed or with heterogenous variances between the groups were stepwise log- or rank-transformed. One-way analysis of variance (ANOVA) was performed with non-transformed or log-transformed data. The KRUSKAL-WALLIS test was used for rank-transformed data. In case of significant differences (found by ANOVA or KRUSKAL-WALLIS test), inter-group comparisons with the control group were made by parametric or non-parametric DUNNETT multiple comparison tests (p ≤ 0.05 and p ≤ 0.01). Parametrical values not captured by Provantis (e.g. number and weight of the fetuses) were analysed by the DUNNETT test (p ≤ 0.05 and p ≤ 0.01). Prior to the DUNNETT test homogeneity of variances was tested using the BARTLETT test. In case of heterogeneity of variances, the STUDENT's t-test was carried out (p ≤ 0.05 and p ≤ 0.01). Statistical analyses of non-parametrical data (e.g. resorption-, malformation-, variation and retardation rates) were performed using the following settings: FISHERs exact test, n < 100; (p ≤ 0.05 and p ≤ 0.01) or Chi2 test, n ≤ 0.01 (p ≤ 0.05 and p ≤ 0.01). Significantly different data are indicated in the summary tables of the result sections of the report.
Historical control data:
LPT Background Data range of mean values per group (n=56 control or n= 143 test item groups; Data taken from 2000 - 2014) were considered in the evaluation of test data and results.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
At 600 mg Sulphamic Acid/kg b.w./day several dams revealed signs of toxicity in form of behavioural changes and a reduction in the mean body weight and in food consumption in comparison to the control group.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
An increased number of resorptions, a slightly reduced fetal body weight and delayed ossifications for the os ischii, the sacral and caudal vertebral bodies and the metatarsalia were noted at the materno-toxic dose level of 600 mg Sulphamic Acid/kg b.w./day.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: embryotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Examination of the dams:

Mortality: None of the dams died prematurely.

Clinical signs: At 600 mg test item/kg b.w./day 11 of 20 dams revealed several signs of clinical toxicity like piloerection, an extreme yellow discoloured urine, salivation and an decreased or increased water consumption.

Group 4: Dams with signs of toxicity(+: sign observed (.): number of test days)

Dam no.

Piloerection

Extreme yellow discoloured urine

Salivation

Water consumption decreased

Water consumption increased

77

 

 

+ (6)

+ (6)

 

78

 

 

+ (7)

 

+ (3)

79

 

 

 

 

+ (3)

80

 

 

 

 

+ (8)

81

 

+ (7)

+ (7)

+ (9)

+ (1)

82

 

 

+ (4)

 

 

85

 

+ (10)

 

 

+ (10)

86

 

 

+ (3)

 

 

91

+ (7)

+ (8)

 

+ (10)

 

92

+ (2)

+ (4)

+ (9)

+ (9)

 

93

+ (2)

 

+ (6)

 

 

 

body weight gain: Treatment with 600 mg test item/kg b.w./day led to a transient reduction in body weight gain for the dams of the high dose group between gestation day 6 and 9, leading to a reduced body weight by 4.4% (statistically not significant) in comparison to the control group on the day of laparotomy. The net weight change (body weight gain without the uterus) from the start of treatment until laparotomy was decreased by 55.7% for the dams of the high dose group (600 mg test item/kg b.w./day) in comparison to the control group (statistically not significant).

Food consumption: 600 mg test item/kg b.w./day caused a reduced food intake after the start of treatment between gestation days 6 and 12 by max. 19.1% (statistically not significant).

Drinking water consumption: A decreased water consumption was noted during the daily visual appraisal for 4 of 20 dams and an increased water consumption for 5 of 20 dams of the high dose group (600 mg test item/kg b.w./day).

Necropsy findings: No changes were noted during internal inspection at laparotomy.

Uterus and carcass weights: At 600 mg test item/kg b.w./day a slight reduction in the gravid uterus weight by 11.2% (statistically not significant) was noted.

Examination of the fetuses:

An increased number of resorptions was noted in the high dose group (600 mg test item/kg b.w./day), leading to an increased post-implantation loss and a statistically significantly (p ≤ 0.05) increased ratio of resorptions to implantation sites.

Parameter

Mean number of resorption per dam

LPT Background Data

range of mean values per group

(n=56 control or n= 143 test item groups;

Data taken from 2000 - 2014)

Resorptions per dam

Group 1:

0.4

0.0 - 1.8
0.0 - 6.4

(Control)
(test item groups)

Group 2:

0.3

Group 3:

0.4

Group 4:

0.9*

*/**:

(p ≤ 0.05 / p ≤ 0.01) Chi2t-test

 

Parameter

Group 1

Control

(n=20)

Group 2

60 mg/kg

(n=20)

Group 3

200 mg/kg

(n=20)

Group 4

600 mg/kg

(n=20)

 

Corpora lutea

total

per dam

 

284

14.2

275

13.8

281

14.1

277

13.9

 

Implantation sites •1

total

per dam

 

280

14.0

268

13.4

272

13.6

270 #1

13.5

 

Resorptions •2

total

per dam

 

8

0.4

5

0.3

7

0.4

17* #2

0.9

 

Early resorptions •2

total

per dam

 

6

0.3

4

0.2

6

0.3

11

0.6

 

Late resorptions •2

total

per dam

 

2

0.1

1

0.1

1

0.1

6 #1

0.3

 

Live fetuses •3

total

per dam

 

272

13.6

263

13.2

265

13.3

254

12.7

 

Dead fetuses at laparotomy

total

 

0

0

0

0

 

Pre-implantation loss

mean %

 

1.4

2.6

3.1

2.0

 

Post-implantation loss

mean %

 

2.8

1.9

2.8

6.4

 

 

*

Significantly different from the controls at p ≤ 0.05, Chi2test.

 

**

Significantly different from the controls at p ≤ 0.01, Chi2test.

 

 

 

 

#1

including twins

 

#2

mostly due to dam no. 88 with 6 resorptions (1 early + 5 late resorptions) and the number of resorptions per dam is still in the range of background data (0.0 - 1.8).

 

 

 

 

For the statistical analyses of the above mentioned parameters of reproduction the following comparisons were performed:

 

•1

comparing the values of implantation sites/corpora lutea of the test group with the ratio of implantation sites/corpora lutea of the control group using the Chi2test. This comparison is described as pre-implantation loss .

 

•2

comparing the values of resorptions/implantation sites of the test group with the ratio of resorptions/implantation sites of the control group using the Chi2test.

 

•3

comparing the values of live fetuses/implantation sites of the test group with the ratio of fetuses/implantation sites of the control group using the Chi2test. This comparison is described as post-implantation loss.

 

Mortality: No dead fetuses were noted in the study.

Sex distribution: No test item-related differences were noted.

Fetal weights: 600 mg test item/kg b.w./day resulted in a slight reduction in fetal body weight by 5.9%.

 

 Parameter  Weight of the fetuses (g)     LPT Background Data#   

range of mean values per group

(n=56 control or n= 143 test item groups;

Data taken from 2000 - 2014)
 Male and female fetuses together  Group 1:  3.4 3.2 - 4.0  (Control)
   Group 2:  3.5

3.1 - 4.0

 (test item groups)
   Group 3: 3.6 **   3.1 - 4.0  (test item groups)
   Group 4:  3.2  3.1 - 4.0  (test item groups)
Male fetuses   Group 1:  3.5  3.2 - 4.1  (Control)
   Group 2:  3.6  3.1 - 4.2  (test item groups)
   Group 3:  3.6  3.1 - 4.2  (test item groups)
   Group 4:  3.3  3.1 - 4.2  (test item groups)
 Female fetuses  Group 1:  3.3  3.1 - 3.8  (Control)
   Group 2:  3.4  3.0 - 3.9  (test item groups)
   Group 3:  3.5 **  3.0 - 3.9  (test item groups)
   Group 4:  3.1  3.0 - 3.9  (test item groups)
             #: not audited by QAU
             */**: (p ≤ 0.05 / p ≤ 0.01) Dunnett or Student's t-test

Placental weights: No test item-related differences were noted.

Runts: 2 runts were noted in the control group and no runts in the treatment groups.

Malformations: No test item-related malformations were noted in the fetuses during external / internal examination, skeletal examination (according to DAWSON) and during soft tissue evaluation (according to WILSON).

Variations: No test item-related variations were noted in the fetuses during external / internal examination, skeletal examination (according to DAWSON) and during soft tissue evaluation (according to WILSON).

Skeletal examination of the foetuses: No malformations were noted during skeletal examinations of the fetuses according to DAWSON in the control group and in any of the treatment groups.

Unclassified external observations: The observation of twins (5-1 and 5-2) from dam no. 92 of the high dose group (600 mg Sulphamic Acid/kg b.w./day) was considered as an unclassified external observation.

Retardations: At 600 mg test item/kg b.w./day the examination of the skeleton according to DAWSON revealed test item-related increased fetal incidences (statistically significant at p ≤ 0.05 or p ≤ 0.01) for the following parameters: absence of ossification in metatarsalia 2-5, caudal vertebral bodies (all bodies unossified), os ischii incompletely ossified or unossified, sacral vertebral body/bodies unossified.

Parameter

Group values observed in this study

(fetal incidences in %)

LPT Background Data#

range of mean values per group

(fetal incidence in mean %)

(n=55 control or n= 141 test item groups;

Data taken from 2000 - 2014)

Absence of ossification in metatarsalia

Group 1:

8.8

0.0 - 42.3
0.0 - 37.6

(Control)
(test item groups)

Group 2:

4.5

Group 3:

9.0

Group 4:

18.9*

Caudal vertebral bodies, all bodies unossified

Group 1:

16.2

0.0 - 37.4
0.0 - 29.6

(Control)
(test item groups)

Group 2:

22.7

Group 3:

16.5

Group 4:

40.9**

Os ischii incompletely ossified

Group 1:

2.2

0.0 - 2.5
0.0 - 5.3

(Control)
(test item groups)

Group 2:

5.3

Group 3:

1.5

Group 4:

7.1*

Os ischii unossified

Group 1:

7.4

0.0 - 14.0
0.0 - 7.6 

(Control)
(test item groups)

Group 2:

7.6

Group 3:

6.0

Group 4:

18.1**

Sacral vertebral body/bodies unossified

Group 1:

6.6

0.0 - 20.8
0.0 - 12.4

(Control)
(test item groups)

Group 2:

8.3

Group 3:

7.5

Group 4:

23.6**

Hyoid unossified

Group 1:

71.3

0.0 - 89.4
0.0 - 93.7

(Control)
(test item groups)

Group 2:

64.4

Group 3:

56.4**

Group 4:

59.8*

Skull incomplete ossification

Group 1:

71.3

1.4 - 71.3
1.4 - 79.5

(Control)
(test item groups)

Group 2:

79.5

Group 3:

59.4*

Group 4:

74.8

Sternebra(e) incompletely ossified

Group 1:

7.4

5.5 - 86.2
0.0 - 91.4

(Control)
(test item groups)

Group 2:

17.4**

Group 3:

16.5*

Group 4:

11.0

Sternebra(e) unossified

Group 1:

91.9

5.8 - 91.9
1.2 - 88.5

(Control)
(test item groups)

Group 2:

84.8

Group 3:

80.5**

Group 4:

86.6

Thoracic vertebral body/bodies dumbbell-shaped

Group 1:

27.9

0.0 - 27.9
0.0 - 43.2

(Control)
(test item groups)

Group 2:

43.2**

Group 3:

36.8

Group 4:

29.9

#:

not audited by QAU

*/**:

(p ≤ 0.05 / p ≤ 0.01) Fisher or Chi2- test

The statistically significant changes listed in the table above are not considered to be test item-related due to the following reasons:

Hyoid unossified, skull incomplete ossification, sternebra(e) unossified: A decreased incidence of unossified parts of the skeletal in comparison to the control group is not an adverse effect.

Sternebra(e) incompletely ossified, thoracic vertebral body/bodies dumbbell-shaped: The increase in the fetal incidence is restricted to the low and/or the intermediate dose group, whereas no statistically significant differences between the high dose group and the control group were noted.

No malformations or test item-related variations were noted during the external macroscopic examination, the macroscopic internal examination for gross changes of internal organs, the skeletal examination according to DAWSON and the soft tissue examination according to WILSON for the fetuses of the dams treated with 60, 200 or 600 mg Sulphamic Acid/kg b.w./day, p.o.

At the materno-toxic dose level of 600 mg Sulphamic Acid/kg b.w./day, p.o. the skeletal examination according to DAWSON revealed retardations in the form of delayed ossifications of the os ischii the sacral and caudal vertebral bodies and the metatarsalia. Delayed ossification has been reported as related to maternal toxicity, especially in case of the phalanges, sternebrae nos. 5/6, the cervical, thoracic, sacral and caudal vertebral centra and the calvarium (Carney and Kimmel, 2007).

 

 

 Number of

 fetuses with

 malformations

 

Group 1

Control

 

Group 2

60 mg

Sulphamic Acid/kg

 

Group 3

200 mg

Sulphamic Acid/kg

 

Group 4

600 mg

Sulphamic Acid/kg

external malformations

 fetal incidence N

                       %

 

1

0.4

 

0

0.0

 

0

0.0

 

1

0.4

external malformations

 litter incidence N

                       %

 

1

5.0

 

0

0.0

 

0

0.0

 

1

5.0

internal malformations

 fetal incidence N

                       %

 

0

0.0

 

0

0.0

 

0

0.0

 

0

0.0

internal malformations

 litter incidence N

                       %

 

0

0.0

 

0

0.0

 

0

0.0

 

0

0.0

skeletal malformations

 fetal incidence N

                       %

 

0

0.0

 

0

0.0

 

0

0.0

 

0

0.0

skeletal malformations

 litter incidence N

                       %

 

0

0.0

 

0

0.0

 

0

0.0

 

0

0.0

soft tissue malformations

 fetal incidence N

                       %

 

0

0.0

 

0

0.0

 

0

0.0

 

0

0.0

soft tissue malformations

 litter incidence N

                       %

 

0

0.0

 

0

0.0

 

0

0.0

 

0

0.0

external variations

 fetal incidence N

                       %

0

0.3

  

0

0.0

0

0.0

 

0

0.0

external variations

 litter incidence N

                       %

 

0

0.0

 

0

0.0

 

0

0.0

 

0

0.0

internal variations

 fetal incidence N

                       %

 

0

0.0

 

0

0.0

 

0

0.0

 

0

0.0

internal variations

 litter incidence N

                       %

 

0

0.0

 

0

0.0

 

0

0.0

 

0

0.0

skeletal variations

 fetal incidence N

                       %

 

0

0.0

 

3

2.3

 

3

2.3

 

1

0.8

skeletal variations

 litter incidence N

                       %

 

6

30.0

 

2

10.0

 

2

10.0

 

1

5.0

soft tissue variations

 fetal incidence N

                       %

 

0

0.0

 

3

2.3

 

3

2.3

 

1

0.8

soft tissue variations

 litter incidence N

                       %

 

0

00.0

 

2

10.0

 

2

10.0

 

1

5.0

skeletal retardations

 fetal incidence N

                       %

 

136

100.0

 

132

100.0

 

133

100.0

 

127

100.0

skeletal retardations

 litter incidence N

                       %

 

20

100.0

 

20

100.0

 

20

100.0

 

20

100.0

 

Applicant's summary and conclusion

Conclusions:
Based on the findings of this OECD414 developmental toxicity study with sulphamic acid, no developmental toxicity in the absence of maternal toxicity was observed. Thus, sulphamic acid is not considered causing developmental toxicity in rats.
Executive summary:

In this rat embryotoxicity study, the test item sulphamic acid was administered orally to female rats at dose levels of 60, 200 or 600 mg/kg b.w./day from the 6th to 19th day of pregnancy. Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 200 mg sulphamic acid/kg b.w./day for the dams. At 600 mg sulphamic acid/kg b.w./day several dams revealed signs of toxicity in form of behavioural changes and a reduction in the mean body weight and in food consumption in comparison to the control group. Necropsy revealed no changes for the dams of all treatment groups.  

The no-observed-adverse effect level (NOAEL) for the fetal organism was also at 200 mg sulphamic acid/kg b.w./day.

An increased number of resorptions, a slightly reduced fetal body weight and delayed ossifications for the os ischii, the sacral and caudal vertebral bodies and the metatarsalia were noted at the materno-toxic dose level of 600 mg sulphamic acid/kg b.w./day. No dead fetuses, no malformations and no test item-related variations were noted at any of the tested dose levels.