Octanethioic acid, S-[3-(triethoxysilyl)propyl] ester, reaction products with 2-methyl-1,3-propanediol and 3-(triethoxysilyl)-1-propanethiol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Jun 2007 (Date of Project Protocol) - 26 May 2008 (Date of Final Report)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

Directice 96/54 EEC B.7

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

liquid: viscous

Details on test material:

Colour: Light yellow

Test animals

Species:

rat

Strain:

Wistar

Remarks:

HsdRccHan: WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, D-33178 Borchen, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation:
7-9 weeks
- Weight at main study initiation: females: 146-180g, males 180-216g
- Housing: the animals were kept in Marcolon cages on Altromin saw fiber bedding
- Diet: ad libitum, Altromin 1324 maintenance diet for rats and mice (TPF)
- Water: free access to tap water
- Acclimation period: adequate acclimatization period

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature: 22±3°C
- Humidity: 55±10%
- Air changes: 10x / hour
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Method of administration: Gavage of a suspension, using a stomach tube.
Volume of application: The different doses were applied according to body-weight at a volume of 5 mL/kg bw.
The homogeneity of the preparations was visually checked. During the application procedure no obvious separation was detected,

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of stability and homogeneity was performed previously at a GLP-certified laboratory.
Analysis of dose concentration was performed at a GLP-certified laboratory (reported IBACON project 32664100) using a validated HPLC method. All samples were shipped deep frozen and kept frozen until sample preparation was performed.
Each specimen was analysed at least once. Randomly three samples were analysed twice.
.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily, 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Remarks:

Low dose group, LD group

Doses / concentrations 2

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Remarks:

Medium dose group. MD group

Doses / concentrations 3

Dose / conc.:

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

Remarks:

High dose group, HD group

No. of animals per sex per dose:

Main study: 5
Recovery (control and high dose group only): 5

Control animals:

yes, concurrent vehicle

Details on study design:

The highest dose level (1000 mg test item/kg bw) was chosen with the aim of inducing toxic effects but not death or severe suffering. Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and no-observed effects at the lowest dose level (NOEL).

The animals were randomly assigned to the dose/control groups according to a table of randomized numbers for coding and caged individually.

The study included recovery groups (control and high dose level only). These groups were dosed like the animals from the main study, but after dosing period the animals were left untreated for 14 days before sacrifice.

Examinations

Observations and examinations performed and frequency:

Once before the first exposure and in the final week, detailed clinical observation were made in all animals with specific emphasis on locomotion and behaviour. These observations were made outside the home cage at the same time

CAGE SIDE OBSERVATIONS: Yes
DETAILED CLINICAL OBSERVATIONS: Yes
The observation period was 28 days for the main study and 42 days for the recovery groups. General clinical observation were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. At least twice daily, all animals were observed for morbidity and mortality.
Cageside observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoe, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size.


BODY WEIGHT: Yes
The animals were weighed prior to first application (day 0) and once a week thereafter (main groups: day 7, 14, 21, 27; recovery groups: day 7, 14, 21, 28, 35, 41).
The total and weekly mean weight gain for each group was calculated.
The weight determined on the day of sacrifice were used for the calculation of the relative organ weights.

FOOD CONSUMPTION:
Food consumption was calculated weekly (main groups: day 7, 14, 21, 27; recovery groups: day 7, 14, 21, 28, 35, 41). The total mean and daily food consumption was determined for each group.

HAEMATOLOGY and CLINICAL BIOCHEMISTRY: Yes
The withdrawal of blood was performed by puncture of the abdominal aorta of the anaestheized animals after overnight fasting (this was part of the procedure of killing the animals on the day of necropsy).
The blood was collected into small tubes containing EDTA for heamatology, citrate for clotting tests and plain tubes for clinical biochemistry.

Parameters: Hct, Hb, RBC, WBC, Platelet count; APTT / PTT; Differntial leucocyte count; AST (GOT), ALT (GPT), AP, Chol, TP, GLU, Urea, CREA, ALB, Na, K

URINALYSIS: Yes
Urine was collected into plain tubes by puncture of the urine bladder as a part of the necropsy. Parameters: specific gravity, pH, leucocyte count, nitrite, protein, glucose, ketones, uribilinogen, bilirubin and erythocytes

NEUROBEHAVIOURAL EXAMINATION: Yes
Once before the first exposure and in the fourth or fifth exposure week, respectively sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli, and motor activity assessment) was conducted.
The functional observation battery was designed according to the method described by Moser et al., 1991.

Sacrifice and pathology:

PATHOLOGY:
All animals in the study were subjected to a full, detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

ORGAN WEIGHT:
Liver, kidneys, adrenals, testes, epididymides, thymus, spleen, brain, heart
The relative organ weight was calculated in relation to the bodyweights determined on the day of sacrifice and was expressed in %.

HISTOPATHOLOGY:
Full histopathology was carried out on the preserved organs and tissues of all animals in the Control and High Dose groups (including all gross lesions, brain (representative regions including cerebrum, cerebellum and pons), spinal cord, liver, kidneys, adrenals, stomach, small and large intestines, thymus, thyroid, spleen, lung and trachea, heart, gonads, accessory sex organs (uterus, prostate, vesicula seminalis), urinary bladder, lymph nodes, peripheral nerve and bone marrow).
Additionally, jejunum and ileum were evaluated in all males and females of the intermediate and the recovery groups.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

All rats treated with the test item survived throughout the test period and showed only mild clinical-toxic effects.
Salivation was recorded for almost all animals of the HD group, as well as for three animals of the female MD group.
No other specific findings were noted upon daily clinical observation

Mortality:

no mortality observed

Description (incidence):

All animals survived until scheduled necropsy.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Female: Weight gain of the MD and HD was within the range according to the standard curve for this strain. Weight gain of the female control- and LD animals was slightly beneath the standard growth curve, but still within the range when compared to historical data.

Male: slightly diminished weight gain for control- and HD animals when compared to the standard growth curve. Compared to the historical data, weight gain of all animals was within the range.

As all values were within the range of historical data, toxicological relevance could not be concluded. No differences were detected for the recovery groups.

The results are included below in tabular form.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

All animals showed normal food intake. For the male LD and MD groups a slightly higher food consumption was noted compared to the control animals. This finding correlates with the higher weight gain of those groups.

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

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No severe changes in haematology values (Hct, Hb, RBC, WBC) were found in the main study or recovery animals. No elevation to test article was detected. With the exception of single borderline values, almost all individual and mean values were within the biological ranges. Single deviations were considered to be incidental and toxicological relevance could not be concluded.
With exception of one borderline low value in the female control group and one in the female MD group, all mean and individual Hb values are within the biological range (13.5-18.0 g/dl).

Description (incidence and severity):

All mean and individual AST values are within the biological range (20-110 U/l).
The animals of the male HD group reveal slightly higher values compared to the corresponding control group, reachung statical significance.

All mean and individual ALT values are within the biological range (13-57 U/l). Significant differences are found (on the 95% confidence level) for the male and female HD group.
Although all values are within the biological range a slight dose-dependent rise is noticeable.

With the exception of single low value in the female groups (also in the control), all mean and all individual AP values are within the biological range (48-274 U/l for females, 48-323 U/l for males).
The calculated significance for the female HD group does not correspond with the toxicological significance, as all values are within the biological range.

for the female Control group four CHOL values are found to be slightly below the biological range (0.65-2.21 mmol/L). Also for the female LD and MD groups low borderline values are found. In the female HD group all values are within the biological range. The calculated significance for this female dose group does not correspond with toxicological significance, as it is no marked deviation per se and the calculation is based on the low values of the control animals.
For the male animals all mean and individual values are within the biological range and therefore the calculated significance for the HD group does not correspond to toxicological significance.

Single borderline low TP values are found throughout all male dosage groups, as well as in the female control group.
No siginificant differences are found for any of the groups (on the 95% confidence level). No toxicological relevance is concluded.

Most GLU values of male group animals are at the upper limit, or slightly above the biological range (5-10 mmol/l). Dose dependency is not observed.
The calculated significance for the female LD group does not correspond to toxicological significance, as almost all individual values of the female animals are within the biological range with the exception of one borderline high value in each dose group.

All mean and idividual UREA values (one exception) are within the biological range (4-14 mmol/l).
No significant difference is found (on the 95% confidence level) for any of the groups.

For the female animals 2 borderline low values are found in the control- and one in the MD group. The female LD group reveals one borderline high CREA value and animal no. 46 shows a value clearly above the biological range (31-57 µmol/l). For the male groups one borderline high value is detected in the MD group. No significant differences are found (on the 95% confidence level)

With the exception of 2 borderline ALB low values in the female control- and one in the female HD group, all individual ALB values of the female animals are within the biological range (28-41 g/l).
For the male animals one borderline low value is found in the MD group. All other individual values are within the biological range.
Significant difference is found (on the 95% confidence level) for the female LD and MD groups. Toxicological relevance is not concluded as most ALB values are within the biological range.

All mean and individual Na values are within the biological range (136-156 mmol/l). No significant difference is found (on the 95% confidence level).

All mean and individual K values are within the biological range (3-6.5 mmol/l). No significant difference is found (on the 95% confidence level).

Urinalysis findings:

no effects observed

Description (incidence and severity):

No relevant differences between test and control groups were found. No dose dependency was observed.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No differences were observed concerning functional and behavioural examination prior to application and during the last week of dosing, respectively. No abnormalities were recorded concerning posture, gait, palpebral closure, lacrimation, piloerection, arousal and vocalization.
For supported and unassisted rears no abnormalities were detected. Responses to reflex testing were normal in all groups.

Immunological findings:

not specified

Description (incidence and severity):

Liver:
The mean absolute liver weight is slightly increased in the male and female MD group, as well as in the female HD group, reaching statistical significance for those groups (p>0.05). The mean relative liver weights are slightly increased in the female HD and the male MD group, leading to statistical significance for these groups.
Spleen:
The mean absolute and relative spleen weights are increased in the female MD and HD groups compared to the corresponding control group, resulting in statistical significance for these groups (p<0.05). There are no significant findings for the male groups.
Kidneys:
The mean absolute kidney weights in the female dose groups are slightly increased compared to those in the corresponding Control group, reaching statistical significance (p<0.05) for the MD group. There are no findings for the relative kidney weights and no significant results for the male groups.
Adrenals:
The mean relative adrenal weights in the male LD group are decreased compared to the weights in the corresponding Control group, reaching statistical significance (p<0.05). There are no findings for the females or any ohter male group.
Thymus:
There are no significant results in relative and absolute organ weight for both sexes and any of the groups.
Heart:
The mean relative heart weights in the male MD and HD groups are slightly decreased compared to the weights in the corresponding Control group, reaching statistical significance (p<0.05). There are no findings for the females or other male groups.
Brain:
There are no significant results in relative and absolute organ weight for both sexes and any of the groups.
Testes:
There are no significant results in relative and absolute organ weight.
Epididymides:
There are no significant results in relative and absolute organ weight.

The results are attached below in tabular form.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormal findings were noted at terminal sacrifice in any of the animals.

Description (incidence and severity):

Test item-related histopathological changes were restricted to the small intestine. At termination of the treatment period, lacteal distension was observed in the jejunum in a dose-related manner in the majority of males and females treated at 400 mg/kg/day and in all animals treated at 1000 mg/kg bw/day (details given below).
In a single HD male, minimal lacteal distension was additionally noticed in the ileum.
After the 2-week treatment-free period, the jejunal change was found not to have regressed in males and females in the HD group.
Few other histopathological findings seen at terminal sacrifice. All of them were considered to be incidental in origin and/or within the range of expected changes for rats of this age and strain kept under laboratory conditions.

Details on results:

Evaluation of the data

All animals treated with the test item survived throughout the test period. Salivation was recorded for almost all animals of the HD group, as well as for three animals of the female MD group. No other specific findings were noted upon daily clinical observation. Slightly higher weight gain was observed in the female MD and HD groups, as well as for the male LD and MD group when compared to corresponding controls. As all values were within the range of historical data, toxicological relevance could not be concluded. No differences were detected for the recovery groups. All animals showed normal food intake. For the male LD and MD groups a slightly higher food consumption was noted compared to the control animals. This finding correlates with the higher weight gain of those groups.

No severe changes in haematology values (Hct, Hb, RBC, WBC) were found in the main study or recovery animals. With the exception of single borderline values almost all individual and mean values were within the biological ranges. Single deviations were considered to be incidental and toxicological relevance could not be concluded. No treatment-related macroscopic organ changes were observed at terminal or recovery sacrifice.

The mean absolute and relative liver weights were increased compared to the controls (males: 117%, 112% and 106.6% (105%, 107% and 105%) for the absolute (relative) increase compared to controls for the LD, MD and HD groups, respectively; females: 104%, 109% and 113% (104%, 104% and 107%) for the absolute (relative) increase compared to controls for the LD, MD and HD groups, respectively). In males, no dose-response relationship is found. Furthermore, in absence of histopathological effects this observation is concluded not toxicologically relevant. In females, the effect on liver increases with increased dose (both absolute and relative to bw). As histopathological evaluation did not reveal any changes in the liver, this finding may be interpreted as an adaptive change. This is supported by the fact that no liver effects were found in the recovery group.

Test item-related histopathological changes were restricted to the small intestine. At termination of the treatment period, lacteal distension was observed in the jejunum in a dose-related manner in the majority of males and females treated at 400 mg/kg bw/day and in all animals treated at 1000
mg/kg bw/day. In a single HD male, minimal lacteal distension was additionally noticed in the ileum.

After the 2-week treatment-free period, the jejunal change was found not to have regressed in males and females in the HD group.

Few other histopathological findings seen at terminal sacrifice. All of them were considered to be incidental in origin and/or within the range of expected changes for rats of this age and strain kept under laboratory conditions.

Effect levels

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Formulation analysis:

The recovery rate obtained for analysis of the formulations were low and the analytical method was concluded not to be applicable.

Table 1: Mean weight gain (Day 0-27)

 

Dose group	Sex	Absolute weight gain (g)	Relative weight  gain (%)
Control	Male	86.00	100
	Female	31.20	100
LD	Male	107.00	125
	Female	37.00	119
MD	Male	99.60	116
	Female	43.20*	138
HD	Male	81.60	95
	Female	45.80*	147

 

* Significant (p<0.05)

Historical control range: weight gain of 90-100 g for males and 40-55 g for females

 

Table 2: Small intestinal changes at sacrifice

 

Sex	Males				Females
Number of animals	5	5	5	5	5	5	5	5
Dose group (mg/kg bw/day)	0	150	400	1000	0	150	400	1000
Jejunum: lacteal distension
Minimal	0	0	2	1	0	0	4	0
Mild	0	0	1	4	0	0	0	3
Moderate	0	0	0	0	0	0	0	2
Ileum: lacteal distension
Minimal	0	0	0	1	0	0	0	0

 

 

 

Applicant's summary and conclusion

Conclusions:

A sub-acute repeated dose study was performed according to OECD/EC guidelines and GLP principles. Based on histopathological effects in the jejunum (lacteal distension) in males and females at 400 mg/kg bw/day and above, the NOAEL was concluded to be 150 mg/kg bw/day.