Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15.04.2016 to 08.12.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Specific details on test material used for the study:
Test item content is 56.8% (the remaining content is crystallization water and approx. 0.5% sodium chloride). Dosing was based on anhydrous TMT. A correction factor of 1.76 was used.

Test animals

Species:
rat
Strain:
other: CD / Crl:CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS:
- Strain: Rat / CD® / Crl:CD(SD)
- Number and sex: 100 females
- Breeder: Charles River Laboratories
- Body weigth: 186.9 - 274.0 g on day 0 of pregnancy
- Age: 61 days on day 0 of pregnancy
- Adaptation period: 5 days

ENVIRONMENTAL CONDITIONS:
- Diet: a certified commercial diet (ssniff® R/Z- V1324) ad libitum
- Water: tab water ad libitum
- Housing singly in MAKROLON cages (type III plus)
- Temperature: 22°C ± 3°C
- Rel. Humidity: 55% ± 15%
- Light/dark period: 12/12h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
Tap water was used.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Range of actual/nominal concentration:
95.9% - 105.6% (before administration to the last animal on gestation day 6)
97.5% - 107.2% (before administration to the last animal on the end of the dosing period)
Details on mating procedure:
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.
Duration of treatment / exposure:
Day 6 to 20 of gestation
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 dams per dose, 20 litters were evaluated per dose
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
Individual animals were observed daily for behavioural changes, reaction to treatment, or illness. 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 day. Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. Animals showing signs of abortion or premature delivery would have been sacrificed on the same day.
The weight of each rat was recorded on day 0 of gestation (the day of detection of a positive mating sign), followed by daily weighing - always at the same time of the day.
The quantity of food consumed by each rat was recorded daily. Daily monitoring by visual appraisal of the drinking water bottles was maintained throughout the study.

On gestation day 21, the rats were laparotomised under CO2 narcosis. The ovaries and the uteri of the dams were removed; the gravid 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 sacrifice 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.

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
- distributions in the uterine horns
- absolute number per group
- mean per group
- early resorptions < 2 mm
- late resorptions > 2 mm

Weight of placentae
- individual data per fetus
- mean per litter
- mean per group
- mean per sex and group
Ovaries and uterine content:
On gestation day 21, the rats were laparotomised under CO narcosis. The ovaries and the uteri of the dams were removed; the gravid uteri (in toto) were weighed.
Fetal 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 (location in the uterus and the assignment of the fetuses) 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.
(f) 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).
(g) All fetuses (dead and alive) were inspected externally for damages, especially for malformations.
(h) The fetuses were sacrificed by an ether atmosphere.
(i) Examination of fetuses and determination of number and kind of retardations, variations or malformations:
1) 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).
2) 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.

EVALUATION PARAMETERS:
Weight of fetuses
- individual data per fetus (alive and dead)
- mean per litter
- mean per group
- mean per sex and group

Fetuses
- number per dam (alive)
- number per dam (dead)
- number of fetuses (alive + dead) per sex and dam
- 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

Runts
- number per dam
- number per group

Malformed fetuses, fetuses with variations and with retardations
- type of malformation
- individual data per fetus
- number and incidence (%) per group and litter

- male/female ratio (alive + dead)

Runts
- number per dam
- number per group

Malformed fetuses, fetuses with variations and with retardations
- type of malformation
- individual data per fetus
- number and incidence (%) per group and litter
Statistics:
Parametrical data:
The statistical evaluation of the parametrical values was done by Provantis using the following settings: Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or nonnormality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

Non-parametrical data:
The statistical evaluation of non-parametrical values was done using the FISHER or Chi2 test: 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). The respective calculations for the FISHER and Chi2 test were performed using Provantis (maternal macroscopic findings at necropsy) or an internal computer program (e.g. findings during the fetal skeletal or soft tissue examination).
The statistical evaluation of the pre- and post-implantation index (per group) using the number of corpora lutea, implantation sites and/ or fetuses per group was done using StatXact 4.0.1 software.
Indices:
Calculation of group indices:
Pre implantation loss [%] = (Corpora lutea (per group) - Implantations (per group) / Corpora lutea (per group)) x 100

Post implantation loss [%] = (Implantations (per group) - living fetuses (per group) / Implantations (per group)) x100

Calculation of mean indices per litter:
Pre implantation loss [%] = sum of pre-implantation losses per litter in a group [%] / number of litters in a group

Post implantation loss [%] = sum of post-implantation losses per litter in a group [%] / number of litters in a group

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
At the high dose level (500 mg TMT/kg b.w./day) reduced motility and salivation were noted for all to nearly all animals with an increased severity (slight to moderate in comparison to only slight at the low and the intermediate dose levels). Additionally, animals with a haemorrhagic nose/snout, piloerection, breathing sounds and ptosis were noted.
Mortality:
no mortality observed
Description (incidence):
No premature death was noted in the control group and in the test item-treated groups (50, 200 or 500 mg TMT/kg b.w./day).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
After the start of dosing body weight was slightly retarded for the dams of the high dose group (500 mg TMT/kg b.w./day), leading to slight but statistically significantly reduced body weights on test days 8 and 9 (at maximum 7.8% below the value of the control group on test day 9, see table below). Consequently, compared to the control group, the high dose group (500 mg TMT/kg b.w./day) showed a retarded body weight gain in the 3-day interval after treatment onset (days 6 - 9) leading to a reduced body weight gain in the overall treatment period (days 6 - 21).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
No noteworthy differences were noted between the control group and the low and intermediate dose groups (50 or 200 mg TMT/kg b.w./day). At 500 mg TMT/kg b.w./day slight to moderate reductions in food consumption were noted after start of treatment between gestation days 6 and 9 (at maximum 30.0% below the value of the control group).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test item-related changes in drinking water consumption were noted between the dams of the control group and the dams of the treatment groups by visual appraisal.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The heart, the liver and the kidneys were weighed and no test item-related differences for the absolute and the relative organ weights were noted between the control group and the low and intermediate dose groups (50 mg and 200 mg TMT/kg b.w./day).
Regarding the absolute organ weights, an increased weight of the left and right kidney (+11.9% and +11.3%) was noted in the high dose group (500 mg TMT/kg b.w./day) in comparison to the control group. Furthermore, an increase in the relative organ weights compared to the control group was noted for the liver (+13.0%) of the intermediate dose group (200 mg TMT/kg b.w./day) and for the left and right kidney (+19.7% and +19.0%) as well as for the liver (+11.4%) of the high dose group (500 mg TMT/kg b.w./day) (see table below).
Since the absolute organ weights of the liver did not differ from the treatment groups compared to the control group, the noted increase of the relative organ weights in the intermediate and the high dose group (200 and 500 mg TMT/kg b.w./day) is considered to be spontaneous and not test item-related. In the high dose group (500 mg TMT/kg b.w./day) a slight but statistically significantly increased absolute and relative weight was noted for the left and right kidney. This is regarded to be test-item related.
No test item-related differences were noted between the gravid uterus weight and carcass weight of the control dams and the dams of the treatment groups (50, 200 or 500 mg TMT/kg b.w./day).
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
In the high dose group (500 mg TMT/kg b.w./day) a statistically decreased pre-implantation loss (3.5% vs. 1.0%) and a statistically increased post-implantation loss (2.9% vs. 9.6%) was noted. The decrease of pre-implantation loss was considered to be spontaneous because the implantation was before application of the test item. Though the value of postimplantation loss was slightly above the background range (see table below), this was not considered as test item-related, as it was mostly due to one dam with a total implantation loss (100% or 14 resorptions) (see table below).
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
One dam of the high-dose group had a total implantation loss (100% or 14 resorptions).
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
The placental and fetal weights showed no test item-related differences between the control group and the treatment groups (50, 200 or 500 mg TMT/kg b.w./day).
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Description (incidence and severity):
No test item-related differences between the ratio of male and female fetuses (range: 0.94 - 1.14) were noted between the control group and the treatment groups (50, 200 or 500 mg TMT/kg b.w./day).
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Two runts were noted in the control group and one runt was noted in the intermediate dose group. This is regarded to be within the normal range of variation.

No macroscopically visible external alterations (malformations or variations) were noted for the fetuses of the low and intermediate groups (50 and 200 mg TMT/kg b.w./day) during the macroscopic inspection at laparotomy.
Two fetuses of the high dose group (500 mg TMT/kg b.w./day) were noted with a domed head which is classified as variation. Both fetuses did not reveal any correlated alterations in the skeletal or soft tissue examinations.
A comparison to background data of the institute revealed that this incidence is slightly above but in the range of background data (1 fetus with a domed head in a control group; incidence 0.4). However, if the occurrence of 1 fetus per group is in the range of the spontaneous
variability, it is most likely that also the occurrence of 2 fetuses per group is a spontaneous event. Therefore, the observation of 2 fetuses per group, though it is above the institute's background range, is considered as spontaneous.
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
No skeletal malformations were noted for the fetuses of the control group and the test item-treated groups (50, 200 or 500 mg TMT/kg b.w./day) during the skeletal examination according to DAWSON.
Skeletal variations were noted for the ribs (short or wavy), the sternebrae (bipartite, misaligned to a slight degree or misshapen) and the thoracic vertebral bodies (misaligned) in all groups including the control, so that no test item-related increase in the incidence of the observed skeletal variations was noted for the fetuses of the treatment groups (50, 200 or 500 mg TMT/kg b.w./day) (see table below).

Retardations (delayed ossifications) were related to the skull (incomplete ossification of frontal, parietal, interparietal and/or supraoccipital areas), the hyoid (unossified), the sternum (sternebra(e) incompletely ossified, reduced in size or unossified), the thoracic vertebral bodies (bipartite, dumbbell-shaped or reduced in size), the caudal vertebral bodies (no or only one body ossified), the os pubis and the os ischii (incompletely ossified or unossified) and the metacarpalia and metatarsalia (absence of ossification in metacarpalia 2 to 5 and metatarsalia 2 to 5). No test item-related increase in the incidence of skeletal retardations at 50 or 200 mg TMT/kg b.w./day was noted during skeletal examination according to DAWSON.
Retardations with increased incidences in comparison to the control group that were considered as test item-related were noted at the high dose level (500 mg TMT/kg b.w./day) for the observation of incompletely ossified os pubis and os ischii. In detail, the incidence of the incompletely ossified os pubis in the high dose group was pronouncedly increased compared to controls, and also exceeding background data. Due to the distinct increase of this incidence in the high dose group in comparison to the control group, this observation was considered to be test item-related (see table below).
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No malformations were noted for the fetuses of the control group and the fetuses of the treatment groups (50, 200 or 500 mg TMT/kg b.w./day) during the soft tissue examination according to WILSON.
During the examination of the organs and tissues according to WILSON variations were noted for the cerebral ventricle (dilatation of the 4th ventricle), the kidneys (uni- or bilateral dilatation of the renal pelvis, malpositioned) and the liver (haemorrhagic focus/foci). However, no test item-related differences and no statistically significant differences in the incidences of the observed variations were noted between the control group and the test item-treated groups (50, 200 or 500 mg TMT/kg b.w./day).
Details on embryotoxic / teratogenic effects:
No malformation and no test item-related variations were noted during the macroscopic inspection at laparotomy (including an external inspection and a gross inspection of the organs), the skeletal examination according to DAWSON and the soft tissue examination according to WILSON).
Test item-related retardations (delay in ossification) in form of an incompletely ossified os ischii as well as os pubis were noted at the high dose level (500 mg TMT/kg b.w./day). The low and intermediate dose groups (50 and 200 mg TMT/kg b.w./day) did not show any test item-related skeletal retardations.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: skeletal retardation

Overall developmental toxicity

Key result
Developmental effects observed:
yes
Lowest effective dose / conc.:
500 mg/kg bw/day (actual dose received)
Treatment related:
no
Relation to maternal toxicity:
developmental effects as a secondary non-specific consequence of maternal toxicity effects
Dose response relationship:
no
Relevant for humans:
not specified

Any other information on results incl. tables

Table: Summary of animals examined

   Control 50 mg/kg   200 mg/kg  500 mg/kg
 Treated dams  25 25 

25 

25 
 Not pregnant dams 0
 Dams without viable fetuses 0
 Dams with early delivery 0
 Not examined dams (spare animals) 5
 Evaluated litters  20 20  20  20 

Table: Statistical differences in body weight and body weight gain (*/** statistically significant p ≤ 0.05/0.01 (ANOVA/DUNNETT test))

  Body weight: GD 8       Body weight: GD 9  Mean body weight gain: GD 0 -21         Mean body weight gain: GD 6 -21           
   Weight in g  % Difference to control  Weight in g  % Difference to control  Gain in g  Gain in %    % Difference to control    Gain in g   Gain in %   % Difference to control
 Control  273.12  277.65  -  173.5  79.0  -  133.5  51.3  -
 50 mg/kg 269.83   -1.2  275.64  -0.7  176.0  79.2  + 1.4  136.7  52.2 + 2.4
 200 mg/kg  263.56  -3.5  269.49 -2.9   161.6  73.3  - 6.8  136.7  48.5 - 6.3
 500 mg/kg  252.64 -7.5*   256.11  -7.8**  152.1  68.7  - 12.3  113.5*  43.5 - 15.0 

Table: Absolute and relative organ weights ( ** Statistically significant at p ≤ 0.01 (ANOVA/DUNNETT test))

 Absolute organ weights      
 Organ  Group  Difference
 kidney left  500 mg/kg  +11.9%**
 kidney right  500 mg/kg  +11.3%**
 Relative organ weights      
 Organ  Group  Difference
 liver  200 mg/kg  +13.0%**
 liver  500 mg/kg  +11.4%**
 kidney left  500 mg/kg  +19.7%**
 kidney right  500 mg/kg  +19.0%**

Table: Reproduction data of the dams

 Parameter     Control

 50 mg/kg

(n=20)

  200 mg/kg

(n=20)

  500 mg/kg

(n=21)#

 Corpora lutea  total  285 295 279  306
   mean per dam  14.3 14.8 14.0  14.6 
 Implantation sites  total  275 291  266 303 
   mean per dam  13.8 14.6  13.3  14.4 
 Resorptions  total  8 14  16  29 
   mean per dam  0.4 0.7 0.8  1.4 
 Early resorptions  total  8

13 

14  27 
   mean per dam  0.4 0.7  0.7  1.3 
 Late resorptions  total  0
   mean per dam  0.0 0.1  0.1  0.1 
 Live fetuses  total  267 277  250  274 
   mean per dam  13.4 13.9  12.5  13.7 
 Dead fetuses  total  0

 Pre-implantation loss [%]

 per group ##1

 3.5

1.4 

4.7 

1.0* 

 

 mean per dam

 3.5

1.4 

4.3 

0.9 

Post-implantation loss [%]

 per group ##2

 2.9

4.8 

6.0 

9.6** 

 

 mean per dam ##3

 3.0

4.6 

6.2 

9.7 

Statistical analyses were performed for the mean values per dam using an ANOVA/DUNNETT test.

# One dam with total implantation loss.

##1 The statistical comparison of the pre-implantation loss per group was done by 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 Chi2 test (*/**: p ≤ 0.05/p ≤ 0.01).

##2 The statistical comparison of the post-implantation loss per group was done by 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 Chi2 test

(*/**: p ≤ 0.05/p ≤ 0.01).

##3 Historical control data from the contract institute for postimplantation loss (mean per dam): 1.2 - 7.6 control groups (n=59); 1.0 - 8.4 test item groups (n=152), data taken from 2000 to July 2015

Table: Skeletal variations

 Skeletal variations

Fetal incidence per group [%]

Fetal incidence per group [%]

(historical control data, mean value +/- SD and range)#1

 Rib(s) wavy

 Control

0.0 

5.18 ± 7.2 (0.0 - 27.9) 

 control groups

 

 50 mg/kg

0.7 

 

 

 200 mg/kg

3.2* #2 

 5.43 ± 9.2 (0.0 - 52.9)

 test item groups

 

 500 mg/kg

 0.0

 

 

 Sternebrae misaligned (severity slight)

 Control

4.5

1.33 ± 2.1 (0.0 - 9.5)

control groups 

 

 50 mg/kg

0.7* #3

 

 

 

200 mg/kg

4.0 

 1.46 ± 1.9 (0.0 - 8.9)

test item groups 

 

500 mg/kg

 5.1

 

 

 Sternebrae misshapen

Control

 3.7

0.01 ± 0.1 (0.0 - 0.7)

control groups 

 

50 mg/kg

 0.0* #3

 

 

 

200 mg/kg

 0.0* #3

 0.05 ± 0.2 (0.0 - 1.6)

test item groups 

 

500 mg/kg

 2.2

 

 

 Total fetal skeletal variations

Control

8.2 

 8.2 ± 8.5 (0.0 - 38.0)

control groups 

 

50 mg/kg

2.2* #3 

 

 

 

200 mg/kg

7.2 

 8.3 ± 9.6 (0.0 - 55.0)

test item groups 

 

500 mg/kg

8.0 

 

 

#1 n=59 control or n=153 test item groups, data taken from 2000 to July 2015; not audited by QAU

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

#2: Considered as spontaneously as no dose-response relationship was noted.

#3: Decreased incidences of unossified parts of the skeleton in comparison to the control

group are considered as spontaneously.

Table: Skeletal retardations

 Skeletal retardations

Fetal incidence per group [%]

Fetal incidence per group [%]

(historical control data, mean value +/- SD and range)#1

 Hyoid unossified

 Control

77.6

27.9 ± 23.5 (0.0 - 89.4)

 control groups

 

 50 mg/kg

66.2* #2

 

 

 200 mg/kg

63.2** #2 

27.9 ± 23.0 (0.0 - 93.7)

 test item groups

 

 500 mg/kg

62.8** #2

 

 

Os ischii incompletely ossified

 Control

0.0

0.41 ± 0.7 (0.0 - 2.5)

control groups 

 

 50 mg/kg

0.0

 

 

 

200 mg/kg

0.0

 0.41 ± 1.0 (0.0 - 5.3)

test item groups 

 

500 mg/kg

 2.9* #3

 

 

Os pubis incompletely ossified

Control

 8.2

0.27 ± 0.6 (0.0 - 2.4)

control groups 

 

50 mg/kg

 2.2* #2

 

 

 

200 mg/kg

 4.0

 0.30 ± 0.8 (0.0 - 6.9)

test item groups 

 

500 mg/kg

 18.2**

 

 

Sternebra(e) reduced in size

Control

60.4

 39.45 ± 15.0 (9.3 - 66.7)

control groups 

 

50 mg/kg

69.1

 

 

 

200 mg/kg

57.6

 40.27 ± 14.5 (4.5 - 74.1)

test item groups 

 

500 mg/kg

73.0* #3

 

 

#1 n=59 control or n=153 test item groups, data taken from 2000 to July 2015; not audited by QAU

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

#2: Decreased incidences of unossified parts of the skeleton in comparison to the control group are considered as spontaneously.

#3: Considered as spontaneous as the incidences were still in the range of historical control data

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study, TMT did not show any teratogenic potential.
Executive summary:

In this prenatal developmental toxicity study, the test item 1,3,5-Triazine-2,4,6 -Trithione Trisodium Salt (TMT) was administered orally to female rats at dose levels of 50, 200 or 500 mg/kg b.w./day from the 6th to 20th day of pregnancy. Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 200 mg TMT/kg b.w./day for the dams. None of the dams died prematurely.

Changes in behaviour that were of toxicological relevance were only noted at 500 mg TMT/kg b.w./day accompanied with slight reductions in body weight and food consumption. At necropsy, increased organ weights were noted for the left and right kidney at the high dose level (500 mg TMT/kg b.w./day).

The no-observed-adverse-effect level (NOAEL) for the fetal organism was also 200 mg TMT/kg b.w./day. At the materno-toxic dose level of 500 mg TMT/kg b.w./day test item-related retardations were noted in the form of delayed ossifications of the os pubis and os ischii. However, these retardations are considered as a secondary effect. It is wellknown that maternal stress caused by materno-toxic effects, e.g. reduced food consumption, has an influence on skeletal variations.#

The reproductive parameters (number of implantation sites, number of resorptions and number of fetuses) were not influenced by the test item. No dead fetuses, no malformations and no test item-related variations were noted.

Under the conditions of the study, TMT did not show any teratogenic potential.

# Amugongo SK, 2010. In utero Sources of Skeletal Variation: the Role of Maternal Prenatal Stress (Dissertation, University of Berkeley, USA).