Sulfonic acids, C6-8-alkane, sodium salts

Endpoint summary

Description of key information

In a 90 -day repeat dose toxicity study performed according to OECD TG 408 the NOAEL for systemic toxicity was 1000 mg a.i./ kg bw/day.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 September - 17 December 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Batch/Lot number: 0008282300
Description: Clear liquid
Purity: 40.9 % in water
Expiry date: 13 July 2021
Storage conditions: Controlled room temperature (15-25 °C, ≤70% relative humidity)

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI

Details on species / strain selection:

The Wistar rat is regarded as suitable species for toxicology studies and is one of the standard strains for repeat-dose toxicity studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, approx. 6 weeks old at start of treatment.
- Weight at study initiation: Males: 205 – 239 g; Females: 151 – 179 g; did not exceed ± 20 % of the mean weight for each sex at onset of treatment.
- Fasting period before study:
- Housing:
- Diet: Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice –breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany (batch number: 369 53786, expiry date: 29 February 2020 and batch number: 746 55782, expiry date: 30 April 2020), ad libitum
- Water: Tap water from the municipal supply, as for human consumption, from a 500 mL bottle, ad libitum
- Acclimation period: 6 days (males), 7 days (females)

DETAILS OF FOOD AND WATER QUALITY:
The food is considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided analytical certificates for the batchs used, which is archived with the raw data.
Water quality control analysis was performed at least once every three months and microbiological assessment is performed monthly, by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8200 Veszprém, József Attila u. 36., Hungary). The quality control results are retained with the raw data in the archives at Charles River Laboratories Hungary Kft.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.2 – 24.3 °C
- Humidity (%): 20 – 64 %
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12 hours light daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: To: 17/09/2019 - 17/12/2019

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected as it is one of the possible routes of human exposure.

Vehicle:

water

Remarks:

distilled

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was formulated at appropriate concentrations in the vehicle (distilled water) as a visibly homogenous formulation in the Pharmacy of Charles River Laboratories Hungary Kft. Formulations were prepared freshly prior to administration on Day 0. On subsequent days, formulations were prepared up to 9 days before use (formulation were kept in sealed containers at room temperature until use). Stability of the test item in the vehicle was assessed under the conditions used on the study during the analytical method validation (19/211-901AN). In that study, formulation samples in the 2-200 mg/mL concentration range (using distilled water as vehicle) were proven as being stable for 10 days when stored at room temperature.
A correction factor of 2.45 was used at dose formulation preparation to take account of the active substance content in the test material.
VEHICLE
- Lot/batch no.: 201906031 / 201909057

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of test item formulations for concentration and homogeneity was performed using an HPLC-MS method at the Test Site 1. Duplicate samples from the top, middle and bottom were taken from test item formulations four times during the study (in the first, fifth, tenth and thirteenth weeks of the treatment of males). One set was used for analysis and one set as a back-up. Similarly, duplicate samples were taken from the middle of the vehicle control formulation for concentration measurement.
Acceptance criteria of the concentration analyses was set according to the analytical method validation, expected to be at 100 ± 10 % of the nominal concentrations.
Acceptance criteria for homogeneity was that the CV of replicates (top, middle and bottom of test item formulations) was less than 10%.

A summary of the analysis results obtained are presented in the results section below (Table 7).
Detailed results from each sampling point are attached.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

based on active ingredient

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

based on active ingredient

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

based on active ingredient

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

No. of animals per sex per dose:

10 male/10 female per dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were set by the Sponsor in agreement with the Study Director, based on a dose range finding study and other available data provided by the Sponsor, with the aim of inducing toxic effects but no death or suffering at the highest dose (1000 mg a.i./kg bw/day). Based on the available data, doses of 100, 300 and 1000 mg a.i./kg bw/day were selected for the main study. The aim was to use a maximum dose of 1000 mg a.i./kg bw/day to induce some toxicity and establish a NOAEL at the lowest dose.
- Rationale for animal assignment: During the acclimation period, the animals were assigned to their respective dose groups by randomisation based on body weights. Animals were randomly allocated to the control and dose groups based on the most recent actual body weight; the software PROVANTIS v.9 was used in order to verify homogeneity/variation among/within groups. Males and females were randomised separately.
- Fasting period before blood sampling for clinical biochemistry: overnight

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality/morbidity; General clinical observations were made at least once a day at approximately the same time with minor variations as practical. No general clinical observations were made on those days when detailed clinical observations were registered.
- Cage side observations checked in table No.1 were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before the first day of treatment (on Day 0 male/female) and weekly thereafter, in the morning and once before necropsy (table 1).

BODY WEIGHT: Yes
- Time schedule for examinations: At randomisation (pre-treatment period), on the first day of treatment (Day 0, prior to start of treatment), then weekly, including on Day 89, and prior to necropsy, fasted on Day 90.

FOOD CONSUMPTION:
- The determination of food consumption was performed for all groups once a week. Food was measured on Day 0, the remaining, non-consumed food was weighed weekly from Day 7 with a precision of 1 g. Weekly and total food consumption were calculated for reporting purposes.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment on Day 1; Day 86
- Dose groups that were examined: All groups on Day 1; Control and high dose group on Day 86

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 90
- Anaesthetic used for blood collection: Yes, pentobarbital
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No.2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 90
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in table No.3 were examined.

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: At termination
- Animals fasted: Yes
- How many animals: All animals

URINALYSIS: Yes
- Time schedule for collection of urine: At the end of the treatment period, prior to scheduled necropsy on Day 90
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table No.4 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 13/14
- Dose groups that were examined: All groups
- Battery of functions tested: landing foot splay, fore/hind grip strength and motor activity assessment. Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted and the general physical condition and behaviour of the animals were was tested. A modified Irwin test was performed. A detailed assessment for neurotoxicity effects were made on the basis of these measurements. Parameters such as: body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex and vocalisation, were evaluated.

IMMUNOLOGY: No

OTHER:
EXAMINATION OF VAGINAL SMEARS
- Prior to necropsy, the oestrous cycle of all females was determined by taking vaginal smears, which was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope, in order to provide information regarding the stage of oestrous cycle at the time of sacrifice and assist in the histological evaluation of oestrogen sensitive tissues.

SPERM EVALUATION
At the scheduled termination, after weighing of the testis and epididymis, one of each organ was collected for sperm evaluation as described below.
Enumeration of homogenisation-resistant testicular spermatids and cauda epididymal sperm reserves were performed. In addition, sperm from the cauda epididymis were collected for the determination of motility and sperm morphology. For measurement of sperm morphology, at least 200 sperm per sample were evaluated from fixed, wet preparations and classified as either normal or abnormal (fusion, isolated heads, misshapen heads and/or tails). For enumeration of spermatids, caudal epididymal reserves and morphology, samples from Control and High-dose males were evaluated.
Sperm motility was evaluated immediately after sacrifice. The percentage of progressively motile sperms was determined subjectively.
The smears for morphology were prepared and stored for later analysis, as were the testes and epididymides for homogenisation-resistant sperm counts. Analysies of these endpoints was conducted on the Control and High dose group.

ORGAN WEIGHTS
- Time schedule for examinations: At necropsy
- Dose groups that were examined:All groups
- organs wieghed - see table 5. Paired organs were weighed together. Absolute organ weights were measured, and organ weights relative to the body and brain weights were calculated and reported.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross pathology was performed on all found dead animals.
Necropsy and macroscopic examination were performed on all animals, at the end of treatment period, on Day 90 (after the sample collection for clinical pathology evaluation).


HISTOPATHOLOGY: Yes (see table 6)
Full histopathology was performed in Groups 1 (Control) and 4 (High dose). In addition, any organs or tissues with macroscopic abnormalities (except minor changes) were subjected to histological examination from all groups.

Statistics:

Statistical evaluation was performed with the statistical program package of SAS 9.2 (within the validated Provantis® system). The following decision tree was applied automatically for statistical evaluation of continuous numeric data.
The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis is the better option when the normality and heterogeneity assumptions implicit in the tests are adequate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach is not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons were performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.
For non-continuous data, the Cochran-Armitage test for trend was applied and the Chi-squared test was used for statistical differences relative to control.
For pathology data (macroscopic and microscopic data) the Cochran-Armitage test for trend was applied, then if appropriate, the Chi-squared test homogeneity test. If significance was plausible based on a user-defined value (0.05), a pairwise test of each treatment group versus the control group was made. If the group size was <5 then Fisher’s Exact Test was used, if the group sizes were bigger then the Chi-squared test was used; identifying differences of <0.05, <0.01 or <0.001 as appropriate.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

A nodule was observed in one female receiving 100 mg a.i./kg/day, but this change was not considered to be treatment-related.
Noisy respiration was observed in one male and one female, and piloerection was seen in one male animal in the High dose group. Noisy respiration may be a common local effect after oral dosing with surfactants because even exceedingly small amounts entering the trachea (e.g. from reflux) can cause local irritation. It is not uncommon after oral dosing with surfactants to have occasional deaths due to reflux or similar respiratory exposure to test item in longer term repeat dose studies.
An isolated occurrence of tonic convulsion was noted in one male in the Mid dose group. As no other clinical signs were associated with this symptom, and as no abnormality was found during the necropsy, this was considered to be incidental and not related to treatment.
A white area on an eye was seen in one male in the Control group.
Summary tables from the study report are attached.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Four males (4003, 4004, 4005, 4007) and two females (4504, 4509) were found dead in the High dose group. Two animals died due to an accident during gavage dosing (4005, 4504). The deaths of the remaining animals were considered to be related to the clinical signs (see above – reflux causing local irritation).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no adverse effects of treatment on body weights or body weight gains
A statistically significant slight decrease in the body weight of High dose females on Day 89 (p<0.05) was seen but was in the normal control range and was not considered to be related to treatment. Body weight gains overall were considered to have been unaffected by treatment. Mean data for the Day 0 and Day 90 bodyweights are presented in Tables 8 & 9 for male and female rats, respectively. Report tables including the mean data at each time point and additionally figures showing these data graphically are attached.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Although a transient, statistically significant decrease was observed in food consumption in the Mid and High dose males between Days 7 to 14, this change did not correspond to impaired body weights, and was therefore not considered to be an adverse effect.
Other sporadic numerical differences were considered to be random differences, unrelated to the treatment, and were therefore not considered to be an adverse effect.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment-related changes compared to pre-treatment were noted at ophthalmoscopy examination.
Corneal injury and erosion, and white area on an eye were observed in one Control male animal (1005).

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were observed in the haematology parameters. The few observed statistically significant differences were considered to be incidental, were not dose-related and were within the historical control ranges. These differences were considered not to reflect an adverse effect of the test item.
The coagulation parameters were considered normal in all study groups. A slight, statistically significant increase in APTT was seen in Mid and High dose females, however these values were within the historical control range, hence it was not considered to be test item related.
Selected haematology and coagulation parameters for males and females, respectively are shown in Tables 10 and 11 below.
Summary data from the study report are attached.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The clinical chemistry parameters were considered normal in all study groups.
Summary data are attached.

Endocrine findings:

no effects observed

Description (incidence and severity):

No test item-related differences in thyroid hormone (T3 or T4) or TSH concentrations were observed. A significant decrease in T4 concentration in males receiving 300 mg a.i./kg bw /day was considered not to be consistent with a dose response, was considered incidental and therefore not test item-related. All groups were considered to be normal.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

The urinary pH in males and females receiving 1000 mg a.i./kg/day was statistically low compared to controls, however the values were within the historical control range.
Other statistically significant differences were considered to be incidental.
Please refer to Tables 12 and 13 below and summary data from the study report, attached.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

At the functional observation battery (FOB), performed at the end of exposure (Week 12/13), there were no changes in animal behaviour, general physical condition, grip strength, motor activity, or in the reactions to different types of stimuli in the either the control or treated groups.
The locomotor activity of all groups showed a normal response, initially high then reducing to a plateau at about 30 minutes. There were a few sporadic (statistically significant) effects at some of the 5 minute intervals but these were not considered to be indicative of a treatment-related effect.
Summary data tables from the study report are attached.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In males receiving 300 mg a.i./kg/day, spleen weight relative to bodyweight, and brain weight (p<0.01) were increased and the absolute epididymis weight (p<0.01) decreased. There was no apparent treatment/dose relationship with the differences seen at 300 mg a.i./kg/day.
In males receiving 1000 mg a.i./kg/day, kidney weights relative to bodyweight and brain (p<0.05) were increased, while absolute brain weight (p<0.05) and absolute testis weight (p<0.05) were significantly low. Body weight-relative testis weight was unaffected. The small apparent differences were considered to be related to the terminal body weight of the animals being slightly below the controls. These values were considered to be normal and not of toxicological significance.
Please refer to Table 14 below.
Summary tables from the study report are attached.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Found dead animals
No treatment-related macroscopic findings were detected at necropsy.
Two animals (4005 High Dose male and 4504 High Dose female) died following mis-dosing. Perforation was seen at the thoracic part of the oesophagus, the lungs were dark red, and in the trachea/thoracic cavity reddish material was present. In one case, the thymus was dark red and dry, and red material was present in the perinasal region too.
In the remaining found dead animals (deaths on Days 27, 28, 73 and 75), findings including the dilatation of the stomach and the intestines, red discoloration of the lungs, thymus, mesenteric lymph node or ovary, small mesenteric lymph node, prostate, seminal vesicles, thymus and spleen, focal thickness of the gastric non-glandular mucosa, raised area in the liver and dilatation of the uterine body and horns were considered as agonal incidental or background. The red discoloration of the lungs may have been related to test substance exposure as very small amounts of surfactant may cause local effects, although this is also a common agonal/postmortem change.
Terminated animals
No test item-related macroscopic findings were detected at necropsy.
Other findings, including discoloration of the lungs, thymus and mandibular lymph node, small coagulating glands, seminal vesicles and epididymis, small, or enlarged and soft testes, pelvic dilatation in the kidneys, brown discoloration of the liver, erosion in the cornea, subcutaneous mass, thickness of the non-glandular gastric mucosa, enlarged thyroid glands and dilatation of the uterine body and horns were considered as incidental and not of toxicological significance.
Summary tables from the study report are attached.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Found dead animals
No test item-related microscopic findings were detected in any animal.
Congestion of the lung was seen in these rats, which a common agonal change, but may be related to local effects of test item, i.e. exposure to the test item.
In animals 4005 and 4504 thymus agonal congestion was seen.
In the remaining found dead animals, agonal congestion of the thymus and ovary, atrophy in the prostate, coagulating gland and seminal vesicles, fibrosis in the tongue, bile duct hyperplasia in the liver, dilatation of the uterine horns and intestines were considered as incidental or background, a decreased cellularity in the spleen, thymus, lymph nodes and bone marrow may be correlated with a treatment induced stress reaction, common in animals with some respiratory distress. Similar microscopic changes were not observed in the animals surviving to termination.
Terminated animals
No test item-related microscopic findings were detected.
Findings, seen in the thyroid/parathyroid gland, kidneys, lungs, liver, lachrymal gland, prostate, testes, epididymis, stomach, thymus, tongue, mammary gland and uterine horns of the animals were without meaningful differences in severity and/or incidence and were considered to be incidental and not of toxicological significance.
Summary tables from the astudy report are attached.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

SPERM SAMPLE ANALYSIS
No treatment-related changes were observed in sperm number, morphology or sperm motility in any of the treated males.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no effects seen at high dose

Critical effects observed:

no

Table 7: Summary of results from analysis of test solutions

Nominal concentration (mg a.i./mL)	Measured concentration* (mg a.i./mL)	Percentage of the nominal concentration (%)
Analytical sampling #1(Sampling: 18 September 2019)
Control	Not detected	-
20	18.58±0.68	92.7
60	57.97±1.54	96.4
200	195.55±8.66	97.6
Analytical sampling #2(Sampling: 16 October 2019)
Control	Not detected	-
20	20.90±0.42	104.3
60	62.49±1.86	103.9
200	210.95±7.53	105.3
Analytical sampling #3(Sampling: 20 November 2019)
Control	Not detected	-
20	18.83±0.83	94.0
60	55.93±0.93	93.0
200	187.24±1.85	93.4
Analytical sampling #4(Sampling: 11 December 2019)
Control	Not detected	-
20	20.17±1.21	100.6
60	60.04±0.65	99.9
200	201.54±9,83	100.6

* with 95% confidence interval

All test item formulations were shown to be homogeneous. The measured concentrations evaluated for each test item-dose group varied between 92.7% and 105.3%. No test item was detected in the control samples. These results were within the acceptable ranges (90% - 110%) and were considered suitable for the study purposes.

 

Table 8: Summary of body weight data for males (day 0, day 90)

Sex: Male Day(s) relative to Start Date		0 mg a.i./kg bw/day	100 mg a.i./kg bw/day	300 mg a.i./kg bw/day	1000 mg a.i./kg bw/day
0	Mean	223.7 R^1	223.9	223.2	222.5
	SD	11.3	9.3	9.0	9.9
	Max	239	238	235	233
	Min	206	210	206	205
	N	10	10	10	6
	%Diff		0.1	-0.2	-0.5
90	Mean	506.5 I^1	494.4	476.0	473.2
	SD	43.5	43.3	58.1	31.3
	Max	581	587	572	520
	Min	457	425	399	446
	N	10	10	10	6
	%Diff		-2.4	-6.0	-6.6

Statistical Test: DecisionTree-VES Transformation: Automatic

1 [I - Automatic Transformation: Identity (No Transformation)]

Table 9: Summary of body weight data for females (day 0, day 90)

Sex: Female Day(s) relative to Start Date		0 mg a.i./kg bw/day	100 mg a.i./kg bw/day	300 mg a.i./kg bw/day	1000 mg a.i./kg bw/day
0	Mean	165.6 I^1	163.8	164.6	162.4
	SD	5.5	8.9	6.8	7.8
	Max	177	176	173	173
	Min	158	151	152	155
	N	10	10	10	8
	%Diff		-1.1	-0.6	-1.9
90	Mean	290.0 I,a^2	292.9	274.1	264.4 d^3
	SD	22.6	22.7	16.0	24.2
	Max	321	323	304	291
	Min	246	253	257	232
	N	10	10	10	8
	%Diff		1.0	-5.5	-8.8

Statistical Test: DecisionTree-VES Transformation: Automatic

1 [I - Automatic Transformation: Identity (No Transformation)]

2 [I,a - Automatic Transformation: Identity (No Transformation), (All Groups) Test: Analysis of Variance p < 0.05]

3 [d - Test: Dunnett 2-Sided p < 0.05]

Table 10: Selected haematology parameters in males

Haematology (males)	Group/Concentration (mg a.i./kg bw/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
Red blood count (M/µL)	8.769	8.247*	8.484	8.645
	Differences from control (%)	-6.0	-3.3	-1.4
	Historical control data	8.07 – 9.97
Haematocrit (%)	44.38	42.31**	42.59*	43.83
	Differences from control (%)	-4.7	-4.0	-1.2
	Historical control data	40.0 – 49.5
Haemoglobin Conc. (g/dL)	14.91	14.27*	14.37	14.80
	Differences from control (%)	-4.3	-3.6	-0.7
	Historical control data	13.6 – 16.8
Notes: *= p<0.05, **= p<0.01; Dunnett two-sided test, Values with significant differences are indicated with bold font.

Table 11: Selected coagulation parameter in females

Haematology (females)	Group/Concentration (mg a.i./kg bw/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
APTT (seconds)	11.20	11.83	12.42**	12.49**
	Differences from control (%)	5.6	10.9	11.5
	Historical control data	10.1 – 14.9
Notes: *= p<0.05, **= p<0.01; Dunnett two-sided test, Values with significant differences are indicated with bold font.

Table 12: Selected urinalysis parameters in males

Urinalysis (males)	Group/Concentration (mg a.i./kg bw/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
pH	8.00	8.00	7.90	7.00**
	Differences from control (%)	0	-1.3	-12.5
	Historical control data	6.00 – 8.00
Urinary leucocytes	0.2	1.1	0.2	1.3**
Notes: *= p<0.05, **= p<0.01; Dunnett two-sided test, Values with significant differences are indicated with bold font.

  

Table 13: Selected urinalysis parameters in females

Urinalysis (females)	Group/Concentration (mg a.i./kg bw/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
pH	8.00	8.00	7.90	7.13**
	Differences from control (%)	0	-1.3	-10.9
	Historical control data	6.00 – 8.00
Urine bacteria	2.7	2.6	2.1	1.5**
Notes: *= p<0.05, **= p<0.01; Dunnett two-sided test, Values with significant differences are indicated with bold font.

 

Table 14: Selected organ weights of male animals

Organ weights (males)	Group/Concentration (mg a.i./kg bw/day)
	Control (0)	Low (100)	Mid (300)	High (1000)
Terminal body weight (g)	506.5	494.4	476.0	473.2
	Differences from control (%)	-2.4	-6.0	-6.6
	Historical control data	432 - 657
Brain (g)	2.277	2.203	2.186	2.143*
	Differences from control (%)	-3.2	-4.0	-5.9
	Historical control data	1.91 – 2.46
Brain/Bodyweight (%)	0.452	0.448	0.464	0.454
	Differences from control (%)	-1.0	2.6	0.4
	Historical control data	0.349 – 0.521
Kidneys (g)	2.942	2.996	3.066	3.232
	Differences from control (%)	1.8	4.2	9.8
	Historical control data	2.32 – 4.22
Kidneys/Bodyweight (%)	0.583	0.605	0.646	0.685*
	Differences from control (%)	3.9	10.8	17.5
	Historical control data	0.507 – 0.761
Kidneys/Brain Weight (%)	129.32	135.59	140.02	150.67*
	Differences from control (%)	4.8	8.3	16.5
	Historical control data	103.111 – 189.908
Spleen (g)	0.790	0.871	0.910	0.822
	Differences from control (%)	10.3	15.2	4.0
	Historical control data	0.59 – 1.52
Spleen/Bodyweight (%)	0.157	0.177	0.192**	0.174
	Differences from control (%)	12.9	22.7	11.0
	Historical control data	0.124 – 0.257
Spleen/ Brain (%)	34.75	35.97	41.59**	38.33
	Differences from control (%)	13.9	19.7	10.3
	Historical control data	29.064 – 64.623
Epididymides (g)	1.694	1.597	1.417**	1.490
	Differences from control (%)	-5.7	-16.4	-12.0
	Historical control data	0.67 – 1.98
Epididymides/ Body weight (%)	0.335	0.323	0.302	0.316
	Differences from control (%)	-3.6	-10.0	-5.7
	Historical control data	0.129 – 0.383
Epididymides/ Brain weight (%)	74.51	72.34	64.89	69.67
	Differences from control (%)	-2.9	-12.9	-6.5
	Historical control data	30.180 – 89.545
Testis (g)	4.357	4.058	4.081	3.898*
	Differences from control (%)	-6.9	-6.3	-10.5
	Historical control data	0.79 – 5.16
Testis/Bodyweight (%)	0.864	0.827	0.869	0.825
	Differences from control (%)	-4.2	0.6	-4.5
	Historical control data	0.152 – 1.032
Testis/Brain weight (%)	191.46	184.61	187.10	182.06
	Differences from control (%)	-3.6	-2.3	-4.9
	Historical control data	35.586 – 230.357
Notes: *= p<0.05, **= p<0.01; Dunnett two-sided test, Values with significant differences are indicated with bold font.

 

Executive summary:

A 90-day repeated dose toxicity study was performed to obtain information on the toxicity of the test item when administered by oral gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (control), 100, 300 or 1000 mg a.i./ kg bw/day for 90 days (OECD TG 408).

The dose levels were based on available data, including the results of a 14-day dose range finding (DRF) study, with the aim of inducing toxic effects but no death or suffering at the highest dose (1000 mg a.i./kg bw/day).

Analysis of the test item formulations for concentration and homogeneity demonstrated that no test item was detected in the control samples and that all formulations were homogeneous and were within ± 10 % of the nominal concentrations.

The in-study examinations included clinical signs, mortality, body weights, food consumption, ophthalmoscopy, neurological assessment (including landing foot splay, grip strength and motor activity assessment), clinical pathology, gross pathology, organ weight measurement and histopathology. Full histopathology was performed in Group 1 (Control) and in Group 4 (High dose).

In summary, daily administration of the test item to Wistar rats for 90 days, under the conditions of this study, resulted in a small number of animals at the High dose having noisy respiration, with associated clinical signs and 4 deaths.

The deaths were considered to be as a result of local effects. Treatment with the test item did not result in any other adverse clinical signs.

Bodyweights, body weight gains, and food consumption of the treated groups were not adversely affected by treatment.

Assessment in a functional observation battery (FOB) revealed no changes in: animal behaviour, general physical condition, grip strength, motor activity, or in the reactions to different types of stimuli in both the control and test groups.

No treatment-related changes were noted at ophthalmoscopy examinations.

No treatment-related findings were seen in the haematology or clinical pathology parameters.

A small change in the urinary pH of males and females receiving 1000 mg a.i./kg bw/day was not considered to be an adverse effect of treatment.

No treatment-related organ weight, macroscopic or microscopic findings were observed.

No treatment-related changes were observed in the sperm number, morphology or motility in any of the treated males, nor in thyroid hormone levels, or in any of the sex organs.

In conclusion, under the conditions of this study, the No Observed Adverse Effect level for systemic toxicity (NOAEL) for the test item is 1000 mg a.i./kg bw/day.

 

This is supported by a range finding study in which the substance was administered daily by oral administration to CD® rats for 14 days. The rats were treated with 100, 300 or 1000 mg a.i./kg bw /day. None of the animals died prematurely. All animals treated with 100, 300 or 1000 mg a.i./kg bw/day revealed salivation immediately after administration that lasted for 10 to 15 minutes on up to 5 test days starting on test day 2.

No test item-related influence was noted on body weight and body weight gain, food and drinking water consumption, haematological and biochemical parameters.

Treatment with 1000 mg a.i./kg bw/day resulted in a slight increase in the relative and absolute weight of the stomach (statistically not significant at p<0.01).

The macroscopic post-mortem examination did not reveal any test item-related changes at any dose level.

Histopathological examination revealed a focal squamous cell hyperplasia with hyperkeratosis in the forestomach mucosa near the borderline to the squamosa of the animals treated with 1000 mg a.i./kg bw/day. Further, a moderate sub-epithelial oedema with mixed cell infiltrations was noted in the forestomach of one high dosed female. The histopathological findings correlated with the increase in the stomach weight.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

04-18 October, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP range finder for GLP guideline repeated dose oral toxicity study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

14-day exposure period, no formulation analysis and restricted examination parameters

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: CD® / Crl: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: males 49 days; females 55 days
- Weight at study initiation: males 235.1-269.2 g; females 194.8 - 230.9 g
- Fasting period before study: not applicable
- Housing: Singly in Makrolon cages.
- Diet: Commercial ssniff® R/M-H V1534 diet (ssniff Spezialdiäten GmbH, 59494 Soest, Germany) ad libitum
- Water: ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Humidity: 55% ± 15% (maximum range)
- Air changes (per hr): no data
- Photoperiod: (12 hrs dark /12 hrs light)

IN-LIFE DATES: From: To: 04-18/10/2012

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The substance was dissolved in tap water to the appropriate concentrations. The substance formulations were freshly prepared every day.

VEHICLE
- Justification for use and choice of vehicle (if other than water): water used
- Amount of vehicle (if gavage):10 mL/kg bw administration volume

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Not applicable

Duration of treatment / exposure:

14 days

Frequency of treatment:

Daily

Remarks:

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

No. of animals per sex per dose:

5 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on available data for similar substances
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable
- Section schedule rationale (if not random): Random

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations (see below)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: At the time of group allocations, on the day of commencement of treatment and once a week thereafter.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At necropsy
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters examined (see below).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Necropsy
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters examined (see below)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see below)
HISTOPATHOLOGY: Yes (see below)

Other examinations:

Not applicable

Statistics:

The test item groups 2 to 4 were compared to the control group 1.
The following statistical methods were used:
Body weight / food consumption /haematology / clinical biochemistry /relative and absolute organ weights:
Multiple t-test based on DUNNETT, C. W. New tables for multiple comparisons with a control Biometrics, 482-491 (September 1964)
(p <= 0.01)
The following limit was used: p = 0.01 ^ t = 3.39 for 16 degrees of freedom
Histopathology:
Exact test of R. A. FISHER (p <= 0.05)

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no deaths during the study. None of the male and female rats treated at 100 mg/kg b.w./day revealed any changes of behaviour or external appearance. All animals treated at 300 or 1000 mg/kg b.w./day revealed slight to moderate salivation starting immediately after administration that lasted 10 to 15 minutes on up to 5 test days starting on test day 2.
BODY WEIGHT AND WEIGHT GAIN
No influence was noted on the body weight, the body weight gain and the body weight at autopsy of the animals treated at 100, 300 or 1000 mg/kg b.w./day compared to the control group.
HAEMATOLOGY
No test item-related changes were noted for the haematological parameters of the male and female animals treated at 100, 300 or 1000 mg/kg b.w./day compared to the control group. An increase in MCHC seen in males at 300 mg/kgbw/day was not considered to be test itemrelated as there was no dose dependence.
CLINICAL CHEMISTRY
No test item-related influence was noted on any of the biochemical parameters examined in the male and female animals treated at 100, 300 or 1000 mg/kg b.w./day compared to the control group.
ORGAN WEIGHTS
No statistically significant test item-related influence was noted on the relative or absolute organ weights of the male and female animals treated at 100 or 300 mg/kg b.w./day compared to the control group.
Treatment at 1000 mg/kg b.w./day resulted in a slight increase in the relative (males: 16%, females: 12%) and absolute (males and females: 10%) weight of the stomach (statistically not significant at p <= 0.01).
GROSS PATHOLOGY
Macroscopic inspection at necropsy did not reveal any changes in the male and female animals treated at 100, 300 or 1000 mg/kg b.w./day.
HISTOPATHOLOGY: NON-NEOPLASTIC
The histomorphological examination of rat organs (restricted to controls and high dose groups) treated at a dose level of 1000 mg/kg b.w./day revealed morphological lesions in the forestomach which are considered to be related to the administration of the test item. The forestomach of the male and female rats treated with 1000 mg/kg b.w./day showed a focal squamous cell hyperplasia with hyperkeratosis in the mucosa near the borderline to the squamosa. Further, a moderate subepithelial oedema with mixed cell infiltrations was noted in the forestomach of one female animal. The histopathological findings correlated with the increase in the stomach weight.
Type, incidence and severity of the minimal to mild lympho-histiocytic or mixed cell inflammatory lesions in various organs were comparable in both groups examined and are considered to be spontaneous organ changes and thus not test item-related.
In all other organs there was no morphological difference between the animals treated at 1000 mg/kg b.w./day and the control group.

Basis for effect level:

other: The findings of this extended dose range-finding study are discussed in detail in CSR Section 5.6.3. (Summary and discussion of repeated dose toxicity)

Remarks on result:

not measured/tested

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A 90-day repeated dose toxicity study was performed to obtain information on the toxicity of the test item when administered by oral gavage to groups of 10 male and 10 female Wistar rats at dose levels of 0 (control), 100, 300 or 1000 mg a.i./ kg bw/day for 90 days (OECD TG 408).

The dose levels were based on available data, including the results of a 14-day dose range finding (DRF) study, with the aim of inducing toxic effects but no death or suffering at the highest dose (1000 mg a.i./kg bw/day).

Analysis of the test item formulations for concentration and homogeneity demonstrated that no test item was detected in the control samples and that all formulations were homogeneous and were within ± 10 % of the nominal concentrations.

The in-study examinations included clinical signs, mortality, body weights, food consumption, ophthalmoscopy, neurological assessment (including landing foot splay, grip strength and motor activity assessment), clinical pathology, gross pathology, organ weight measurement and histopathology. Full histopathology was performed in Group 1 (Control) and in Group 4 (High dose).

In summary, daily administration of the test item to Wistar rats for 90 days, under the conditions of this study, resulted in a small number of animals at the High dose having noisy respiration, with associated clinical signs and 4 deaths.

The deaths were considered to be as a result of local effects. Treatment with the test item did not result in any other adverse clinical signs.

Bodyweights, body weight gains, and food consumption of the treated groups were not adversely affected by treatment.

Assessment in a functional observation battery (FOB) revealed no changes in: animal behaviour, general physical condition, grip strength, motor activity, or in the reactions to different types of stimuli in both the control and test groups.

No treatment-related changes were noted at ophthalmoscopy examinations.

No treatment-related findings were seen in the haematology or clinical pathology parameters.

A small change in the urinary pH of males and females receiving 1000 mg a.i./kg bw/day was not considered to be an adverse effect of treatment.

No treatment-related organ weight, macroscopic or microscopic findings were observed.

No treatment-related changes were observed in the sperm number, morphology or motility in any of the treated males, nor in thyroid hormone levels, or in any of the sex organs.

In conclusion, under the conditions of this study, the No Observed Adverse Effect level for systemic toxicity (NOAEL) for the test item is 1000 mg a.i./kg bw/day.

 

This is supported by a range finding study in which the substance was administered daily by oral administration to CD® rats for 14 days. The rats were treated with 100, 300 or 1000 mg a.i./kg bw /day. None of the animals died prematurely. All animals treated with 100, 300 or 1000 mg a.i./kg bw/day revealed salivation immediately after administration that lasted for 10 to 15 minutes on up to 5 test days starting on test day 2.

No test item-related influence was noted on body weight and body weight gain, food and drinking water consumption, haematological and biochemical parameters.

Treatment with 1000 mg a.i./kg bw/day resulted in a slight increase in the relative and absolute weight of the stomach (statistically not significant at p<0.01).

The macroscopic post-mortem examination did not reveal any test item-related changes at any dose level.

Histopathological examination revealed a focal squamous cell hyperplasia with hyperkeratosis in the forestomach mucosa near the borderline to the squamosa of the animals treated with 1000 mg a.i./kg bw/day. Further, a moderate sub-epithelial oedema with mixed cell infiltrations was noted in the forestomach of one high dosed female. The histopathological findings correlated with the increase in the stomach weight.

Justification for classification or non-classification

The NOAEL from a 90 -day study performed using the substance was 1000 mg a.i./kg bw/day. Classification is not required.