Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1)

Endpoint summary

Description of key information

NOAEL (male and female systemic toxicity) = 100 mg/kg bw/d; OECD 422;  Barraclough, 2018

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 July 2017 - 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Reason / purpose for cross-reference:

reference to other study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: CRL:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Margate, UK
- Females (if applicable) nulliparous and non-pregnant: Not reported, assume yes
- Age at study initiation: 10-11 weeks old
- Weight at study initiation: Males: 288.6 - 391.8 g; females: 173.2 - 235.3 g
- Fasting period before study: No
- Housing: During the pre-pairing phase, animals were housed in groups of up to four by sex and dose group. During the pairing phase, one female was housed with one male from the same dose group until mating was confirmed. Following mating, females were housed individually during gestation, and with their litter during the lactation phase. Males were returned to group-housing after the pairing phase.
During neurobehavioral assessments, animals remained in their home cage, except when placed in the testing apparatus.
Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips or European Softwood bedding during the gestation and lactation phases (Datesand Ltd, Manchester, United Kingdom)
- Diet (e.g. ad libitum): Animals had ad libitum access to VRFI diet (Special Diets Services Ltd, Witham, United Kingdom). Each batch of diet was analyzed for specific constituents and contaminants. No contaminants were present in the diet at levels which might have interfered with achieving the objective of the study.
- Water (e.g. ad libitum): Water from the main tap supply was provided ad libitum via water bottles. The water is periodically analyzed for specific contaminants.
- Acclimation period: Upon arrival, all animals were given a clinical inspection for ill health. Animals were acclimated for at least 14 days prior to initiation of dosing (males) or 7 days prior to initiation of smearing (females).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 30-70 % RH
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: From: 26 July 2017 To: 25 September 2017

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared weekly. The test article was formulated as a suspension in Corn Oil. Formulations were stored at room temperature (15 to 25C) in a sealed container, protected from the light.


- VEHICLE
- Justification for use and choice of vehicle (if other than water): Homogenous stable solutions were prepared in corn oil, a guideline recommended vehicle.
- Concentration in vehicle: Not reported
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): MKBP7039V, MKBZ9899V and MKCC0462
- Purity: Assumed pure corn oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Single 5.0 mL aliquot formulation samples were collected from the main in-life experiment study for each animal group at the respective concentrations (0, 20.0, 60.0 and 100.0 mg/mL) on the 26th July 2017 (week 1 of the in-life phase), 9th August 2017 (week 3 of the in-life phase) and 30th August 2017 (week 6 of the in-life phase) for analysis.

A validated gradient elution reversed-phase high-performance liquid chromatographic method with ultraviolet detection (HPLC-UV) was employed for determination of formulation stability and homogeneity, and concentration analyses of formulations administered during the study.

No test item was observed in the control group samples. Measured concentrations from the test item groups were found to be in the range of 89.2 - 102.3 % of nominal values.

Duration of treatment / exposure:

Males: 42 consecutive days (2 weeks prior to pairing; during pairing; and until the day before necropsy)

Females: 61 days (2 weeks prior to pairing [pre-pairing phase]; during the pairing phase; and until LD 13, inclusive, or 25 days post-coitum for females which did not litter and were sent to necropsy on LD 14 or 26 days post coitum). Some females were not dosed on LD 0 if they were observed to be starting or just completed parturition.

Frequency of treatment:

Daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males and 10 females per treatment group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses selected based on the results of a preliminary range-finder test.
- Rationale for animal assignment (if not random): N/A
- Rationale for selecting satellite groups: N/A
- Post-exposure recovery period in satellite groups: N/A
- Section schedule rationale (if not random): N/A

Positive control:

N/A

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule: Male body weights were recorded once during acclimation, before dosing on the first day of dosing, at weekly intervals, and before necropsy. Female body weights were recorded once during acclimation; before dosing on the first day of dosing; at weekly intervals prior to pairing and until confirmation of mating; on Gestation Day (GD) 0, 7, 14, and 20; and on LD 1, 4, 13 and 14 (prior to necropsy).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g/animal/day: Yes
- The amount of food consumed was determined twice weekly prior to pairing (both sexes) and during the post-pairing phase for males. Daily food consumption was recorded for females from GD 0 to 20 and LD 0 to 13. Consumption was calculated as g/animal/day.

WATER CONSUMPTION: Yes
- Time schedule: The amount of water consumed was determined daily during the pre-pairing phase (both sexes). Consumption was calculated as g/animal/day.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At necropsy
- Anaesthetic used for blood collection: Yes (identity); isoflurane
- Animals fasted: Yes
- How many animals: All

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy
- Animals fasted: Yes
- How many animals: All

URINALYSIS: Yes
- Time schedule for collection of urine: Collected overnight during Week 6
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Weekly
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength / motor activity / alertness / approach response / acoustic startle response / bar test / extensor thrust / pain response / righting reflex

IMMUNOLOGY: Yes / No / Not specified
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:

Locomotor activity was assessed in an automated photocell activity recorder for 30 minutes and was undertaken for five selected animals/sex/group (five males with the highest identification numbers and the first five littered females/group). Assessments were performed during Week 6 of dosing (Post Pairing Day 8) for males and on LD 7 for females.

Activity counts were recorded at 5-minute intervals. The following parameters were determined; total activity / total mobile counts

Five selected animals/sex/group (five males with the highest identification numbers and the first five littered females/group) were observed in the hand and in an arena. Assessments were performed during Week 6 of dosing (Post Pairing Day 12) for males and on LD 7 for females. Quantitative assessment parameters are as follows: hind limb foot splay / fore and/or hind limb grip strength

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

Statistics:

Data for each sex were analyzed separately, unless stated otherwise. Only data collected on or after the first day of dosing were analyzed statistically. Except when otherwise stated, tests were performed using a two-sided risk and were considered significant where P ≤ 0.05. By default, significant results were reported as *P ≤ 0.05, +P ≤ 0.01, and/or #P ≤ 0.001.

See details in attached study report.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Urogenital staining and wet abdominal fur were occasionally recorded during Pre Pairing and Pairing for one or two males administered 300 or 500 mg/kg/day. An observation of vocalizing on handling was noted in one female from each test article treated group from Pre-Pairing Day 4 or 5. One female administered 500 mg/kg/day was noted to have wet fur of the anogenital area and/or abdomen during the pre-pairing phase or GD 7. These observations were attributed to test article.

Incidences of mouth rubbing were noted immediately upon the return to the home cage in up to 4 of 10 females administered 500 mg/kg/day. Salivation was observed from Pre-Pairing Day 8 for some females administered 100, 300, or 500 mg/kg/day and persisted through pairing for occasional females, but no dose response was noted. Salivation was not recorded during gestation or lactation. Salivation was also noted for males administered 100, 300, or 500 mg/kg/day, without a dose response, from Pre-Pairing Day 9, 7, and 10, respectively, and until the end of the pre-pairing, through the pairing, and into the post pairing phases. These observations were attributed to the consistency and palatability of the test article and were considered non-adverse.

All other observations, including fur staining, fur loss, sores/lesions, and tail damage, were considered incidental and not related to the test article as they were also observed in control animals, showed no dose relationship, and/or are commonly observed in animals of this age and strain.

Mortality:

not specified

Description (incidence):

One male administered 500 mg/kg/day was found dead on Study Day 16 (Pairing Day 1). No adverse clinical observation were noted prior to the animal being found dead, and no specific cause of demise could be determined from the tissues examined. The death, therefore, had an uncertain relationship to the test article.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test article-related lower mean body weight gain was recorded for males administered 300 or 500 mg/kg/day. Mean male body weight gain was statistically significantly reduced during the pre pairing phase for males administered 500 mg/kg/day and was 67% lower than controls by the end of the pre-pairing phase. Thereafter, mean body weight gain for males was similar to or slightly lower than controls, but this did not attain statistical significance. Overall, the mean male body weight gain at completion of the study was statistically significantly reduced at 57% of the control value for males administered 500 mg/kg/day. While individual incidences did not attain statistical significance for males administered 300 mg/kg/day, the overall gain for the study showed a statistically significantly reduction (77% of the control value). Mean body weight was unaffected in males administered 100 mg/kg/day, compared with controls.

Body weight gain was also lower for females administered 500 mg/kg/day, when compared with controls during late gestation.

Mean body weight during the pre-pairing, gestation, or lactation phases for females administered 100, 300 mg/kg/day were compared with controls and considered unaffected by the test article.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Lower food consumption was noted for males and females administrated 500 mg/kg/day during the first two weeks of dosing. Food consumption was also noted to be lower for 500 mg/kg/day females during late gestation, when compared with controls.

No effect on food consumption was noted following 100 or 300 mg/kg/day administration.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

A test article related effect on water consumption was noted for animals administered 300 or 500 mg/kg/day.

Mean water consumption was increased for animals administered 300 or 500 mg/kg/day, compared with controls, such that at the end of the pre-pairing phase, male water consumption was 67 or 100% greater than controls, respectively, and female water consumption was 38 or 80% greater than controls, respectively.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Hematology parameters were unaffected by Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

A statistically significant elevation in glucose levels was noted in males administered 500 mg/kg/day, and a statistically significant increase in cholesterol, calcium, and inorganic phosphate levels was noted in males administered 300 or 500 mg/kg/day, compared with control. Cholesterol and glucose levels were also elevated in females administered 300 mg/kg/day.

A statistically significant increase in TSH was noted in males administered 300 mg/kg/day (155 % of control) and animals administered 500 mg/kg/day (188 and 176 % of control) following approximately 6 weeks of dosing. These values fell outside of background control ranges.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Mean urine volume was increased for males administered 300 or 500 mg/kg/day, compared with controls (121 and 86 % of control values, respectively); the increase attained statistical significance for males administered 300 mg/kg/day.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

During the assessments, moderate salivation, piloerection, and changes in body/head posture were observed for males in all test article treated groups, compared with controls. In addition, a dose related increase in ataxia was noted, and high stepping, walking on toes, and slow deliberate movements were noted for animals administered 300 or 500 mg/kg/day, although a decrease in activity was not observed in these groups, compared with controls. A statistically significant increased number of rears were noted on Pre-Pairing Day 6 for males administered 300 or 500 mg/kg/day and Post-Pairing Day 12 for males administered 500 mg/kg/day, compared with controls.

Female changes during the assessments included piloerection and salivation, which were generally dose related and behavior changes predominantly characterized by paw flicking (fore paws). A statistically significant decreased number of rears were noted on LD 1 and 7 for females 300 mg/kg/day, compared with controls. A lower number was also recorded on LD 1 for females administered 500 mg/kg/day; these females were early decedents due to litter loss and, therefore, no data are available for LD 7.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Group mean adjusted liver weights were increased for males administered 300 or 500 mg/kg/day, compared with controls, which generally correlated macroscopically with mottled and/or pale discoloration and/or with the microscopic finding of centrilobular hepatocyte hypertrophy.

Group mean adjusted thyroid weights were increased for males administered 300 or 500 mg/kg/day, compared with controls, which generally correlated macroscopically with dark discoloration and/or with the microscopic finding of follicular cell hypertrophy.

Group mean adjusted kidney weights were increased for males administered 300 or 500 mg/kg/day, compared with controls, which generally correlated macroscopically with mottled and/or pale discoloration and/or with the microscopic finding of nephropathy.

All other organ weight changes, including those statistically significant, were attributed to normal biological variation and were considered not test article related as they were small in magnitude, not dose-dependent, inconsistent between sexes, due to normal inter-animal variability, and/or lacked a microscopic correlate.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

In the liver, mottled and/or pale discoloration were recorded in four males administered 100 mg/kg/day, five males administered 300 mg/kg/day and four males administered 500 mg/kg/day.

In the thyroid, dark discoloration was recorded in one male administered 500 mg/kg/day.

In the kidney, mottled and/or pale were recorded in three males administered 100 mg/kg/day, six males administered 300 mg/kg/day and four males administered 500 mg/kg/day.

In the stomach, mucosal thickening was recorded in two males administered 300 mg/kg/day and one male administered 500 mg/kg/day. Raised focus was recorded in one male administered 500 mg/kg/day.

Small thymus was recorded in one female administered 300 mg/kg/day.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Locomotor activity was unaffected by Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1).

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

A range of Amines-related findings were recorded in the liver, thyroid, kidney, and nonglandular stomach of animals administered 100, 300, or 500 mg/kg/day.

In the liver, centrilobular hepatocyte hypertrophy was recorded in two males administered 100 mg/kg/day, all males and all examined females administered 300 or 500 mg/kg/day. This finding was characterized by the enlargement of the hepatocytes, a ground glass appearance of the cytoplasm, and slight compression of the adjacent sinusoids; was principally localized within the centrilobular areas, but occasionally also extended more diffusely to midzonal and periportal regions in affected animals; and correlated generally with increased group mean adjusted liver weights and the macroscopic finding of dark/mottled discoloration.

In the thyroid, follicular cell hypertrophy was recorded in one male administered 100 mg/kg/day, most males and four females administered 300 mg/kg/day and all males and one female administered 500 mg/kg/day and was characterized by follicular cells with increased amounts of cytoplasm, follicular epithelium that ranged from flat to cuboidal or columnar and follicles with decreased amounts of colloid. This finding correlated generally with increased group mean adjusted thyroid weights and with the macroscopic finding of dark discoloration in one male administered 500 mg/kg/day.

In the kidney, nephropathy was recorded in all examined males administered Amines, which was characterized by multifocal areas of tubular basophilia, inflammatory foci dominated by lymphocytes and/or plasma cells, and granular casts principally located at the corticomedullary junction, but occasionally also in cortical and/or papillary tubules. In addition, hyaline droplets in the cytoplasm of tubular epithelial cells of males administered 100, 300 or 500 mg/kg/day were recorded with higher gradings, compared with concurrent controls. This finding was characterized by focally extensive areas of brightly eosinophilic aggregates of cytoplasmic droplets principally located in the superficial cortex, but multifocally extending towards to the corticomedullary junction. Both of these microscopic findings correlated generally with increased group mean adjusted kidney weights and with the macroscopic finding of mottled and/or pale discoloration.

In addition, in the nonglandular stomach, epithelial hyperplasia and orthokeratotic hyperkeratosis were recorded in one male administered 300 mg/kg/day and four males administered 500 mg/kg/day; these correlated generally with mucosal thickening or the presence of a raised focus, macroscopically

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

urinalysis

water consumption and compound intake

Key result

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

histopathology: non-neoplastic

water consumption and compound intake

Key result

Critical effects observed:

no

Table 4       Group mean achieved dose rates for each treatment group (in mg/kg bw/day)

 

Test Group (mg/kg/day)	Nominal formulation concentration (mg/mL)	Actual formulation concentration achieved (% of nominal)
		Week 1	Week 3	Week 6
Control	0	nd	nd	nd
100	20	95.8	97.3	102.3
300	60	96.9	95.7	96.3
500	100	94.8	95.9	89.2

nd: not detected

 

Table 5      Mean body weights and body weight gains/ losses (n=10 unless stipulated otherwise) (F0)

 

Sex / Day	Bodyweight (g)
	Control	100 mg/kg/day	300 mg/kg/day	500 mg/kg/day
MALES
Predose
Day 1	301.5	317.7	313.0	305.7
Pre-pairing
Day 1	325.1	342.6	339.0	328.4
Day 8	345.1	365.8	355.2	331.6
Day 15	363.1	382.3	370.8	341.0
Change Day 1-8	20.1	23.2	16.2	3.3**
Change Day 8-15	18.0	16.5	15.5	9.3*
Pairing
Day 7	369.2	389.2	372.7	348.6 (n=9)
Day 14	385.1	406.3	385.5	365.1 (n=9)
Change Day 7-14	15.9	17.1	12.8	16.5
Post-pairing
Day 7	400.6	420.8	396.2	374.9 (n=9)
Day 13	407.5	424.2	402.2	378.2 (n=9)
Change Day 7-13	6.8	3.4	6.0	3.3
Overall weight change (pre-pairing Day 1 – post-pairing Day 13)	82.5	81.6	63.2*	47.0**
FEMALES
Predose
Day 8	190.1	189.6	208.6	217.3
Pre-pairing
Day 1	198.1	197.9	208.6	215.2
Day 8	208.6	199.4	212.1	225.4
Day 15	217.3	200.6	209.4	219.4
Change Day 1-8	10.5	10.8	12.8	8.8
Change Day 8-15	8.6	6.5	13.3	10.0
Gestation
Day 0	220.6 (n=9)	242.0	268.4 (n=9)	326.9 (n=9)
Day 7	217.5 (n=9)	238.8	263.4 (n=9)	314.7 (n=9)
Day 14	228.0 (n=9)	247.3	277.1 (n=9)	332.3 (n=9)
Day 20	221.0 (n=9)	244.6	269.1 (n=9)	311.6 (n=9)
Change Day 0-7	21.4	21.3	19.4	23.7
Change Day 7-14	26.4	24.5	29.8	24.4
Change Day 14-20	58.5	51.3	55.2	42.6
Lactation
Day 1	252.8 (n=9)	252.3	255.3 (n=8)	-
Day 4	256.6 (n=8)	259.4	261.6 (n=8)	-
Day 13	275.5 (n=8)	274.9	286.8 (n=8)	-
Day 14	258.2 (n=8)	255.0	254.9 (n=8)	-
Change Day 1-4	3.7	7.1	6.4	-
Change Day 4-13	18.9	15.6	25.2	-
Change Day 13-14	-17.3	-20.0	-31.9*	-
Overall weight change (pre-pairing Day 1 – gestation Day 20)	128.6	116.8	134.0	110.7

* p<0.05, **p<0.01

 

Table 6      Haematology, clinical chemistry, urinalysis and pathology findings (F0)

 

Doses (ppm)	Control	100 mg/kg/day	300 mg/kg/day	500 mg/kg/day	Control	100 mg/kg/day	300 mg/kg/day	500 mg/kg/day
	Male				Female
Haematology
Haemoglobin (g/dl)	14.6	15.2	15.1	14.7	14.8	14.7	14.8	15.5 #
Red blood cells (10^12/L)	8.34	8.56	8.48	8.22	7.77	7.70	7.64	7.93 #
Packed cell volume (%)	45.4	46.9	47.2	45.5	44.1	44.7	44.9	48.5 #
Mean cell volume (fL)	54.5	54.9	55.7	55.3	56.8	58.0	58.9	61.1 #
Mean cell haemoglobin (pg)	17.5	17.7	17.9	17.8	19.0	19.1	19.4	19.6 #
Mean cell haemoglobin conc. (g/dL)	32.2	32.3	32.1	32.3	33.4	33.0	33.0	32.1 #
Reticulocytes (%)	2.5	2.2	2.5	2.4	3.4	3.8	3.7	2.8 #
Absolute reticulocytes (10^9/L)	204.3	189.3	208.6	197.2	261.9	294.6	284.5	218.6 #
Red blood cell distribution (%)	12.5	13.1	13.3	13.6	12.5	12.4	14.5	14.2 #
Haemoglobin distr. width (g/dL)	2.32	2.28	2.29	2.28	1.93	1.91	1.95	1.76 #
White blood cells (10^9/L)	3.6	3.8	4.6	4.6	5.2	4.9	4.3	3.0 #
Neutrophils (10^9/L)	0.75	0.66	0.86	0.77	2.84	2.15	1.81	1.40 #
Lymphocytes (10^9/L)	2.69	2.98	3.56	3.62	2.11	2.46	2.20	1.47 #
Monocytes (10^9/L)	0.05	0.06	0.09**	0.08	0.17	0.17	0.22	0.11 #
Eosinpphils (10^9/L)	0.05	0.06	0.06	0.05	0.05	0.04	0.04	0.04 #
Basophils (10^9/L)	0.00	0.01	0.01	0.01	0.00	0.00	0.01	0.00 #
Large unstained cells (10^9/L)	0.01	0.01	0.03	0.02	0.04	0.0	0.06	0.01 #
Neutrophils (%)	24	20	19	17	56	43	41	46 #
Lymphocytes (%)	73	76	77	80	39	53	52	49 #
Monocytes (%)	2	2	2	2	4	3	5	4 #
Eosinophils (%)	2	2	2	1	1	1	1	1
Basophils (%)	0	0	0	0	0	0	0	0
Large unstained cells (%)	0	0	1	0	1	1	1	0
Platelets (10^9/L)	729	728	771	782	936	932	895	636 #
Platelet crit (%)	0.58	0.59	0.61	0.62	0.61	0.63	0.60	0.40 #
Mean platelet volume (fL)	8.1	8.1	7.8	7.9	6.5	6.8	6.7	6.3 #
Platelet distribution width (%)	55.5	55.7	52.4	54.4	51.9	50.1	49.9	50.2 #
Prothrombin time (sec)	20.1	20.9	21.1	21.1	22.2	22.5	23.7	21.5 #
Thromboplastin time(sec)	16.7	17.2	17.5	16.8	24.0	19.8	18.3	15.7 #
Fibrinogen (g/L)	1.34	1.37	1.42	1.42	2.11	1.93	2.07	1.70 #
Blood chemistry
Aspartate aminotransferase (IU/L)	56	47**	51	46*	111	94	104	62 #
Alanine aminotransferase (IU/L)	38	30	39	42	65	61	75	45 #
Alkaline phosphatase (IU/L)	70	77	84	94	74	65	77	98 #
Total cholesterol (mmol/L)	1.5	1.7	1.9*	2.0**	2.1	2.1	2.6*	2.5 #
Total bilirubin (µmol/L)	<1.7	<1.7	<1.7	<1.7	<1.7	<1.7	<1.7	<1.7
Total protein (g/L)	59	59	59	57	56	56	59	58 #
Albumin (g/L)	39	39	37	37	32	33	35	35 #
Globulin (g/L)	20	21	22	20	25	23	25	23 #
Albumin/Globulin ratio	1.9	1.9	1.7	1.9	1.3	1.5	1.4	1.5 #
Sodium (mmol/L)	142	142	142	141	138	13	138	140 #
Potassium (mmol/L)	3.7	3.6	3.8	3.7	3.6	3.5	3.8	3.8 #
Chloride (mmol/L)	102	102	102	102	95	96	98	99 #
Calcium (mmol/L)	2.49	2.54	2.57*	2.58*	2.69	2.73	2.75	2.78 #
Inorganic phosphate (mmol/L)	1.6	1.6	1.9*	1.9*	3.3	3.4	3.2	3.0 #
Enzymatic creatinine (µmol/L)	27	31	29	30	46	46	38	32 #
Urea (mmol/L)	5.7	5.9	5.5	6.2	16.6	15.6	14.5	10.4 #
Glucose (mmol/L)	8.8	9.9	10.1	11.0**	8.4	8.9	10.2*	7.9 #
Serum bile acids (µmol/L)	14.40	<14.45	<15.66	15.22	38.89	22.33	50.98	12.08 #
Urinalysis
Volume (mL)	12.6	11.5	27.8*	23.4	-	-	-	-
Specific gravity (g/L)	1.020	1.019	1.011	1.015	-	-	-	-
Thyroid hormone analysis
Total T3 (nmol/L)	<0.57	<0.57	<0.54	<0.54	-	-	-	-
Imulite Total T4 (nmol/L)	59	55	57	48	-	-	-	-
Rapid thyroid stim. hormone (µlU/mL)	0.33	0.42	0.84*	0.95**	-	-	-	-
Pathology (remarkable changes)
Adrenals - pale, moderate - large, slight - pale, slight	(n=5) 0 0 0	(n=5) 0 0 0	(n=5) 0 0 0	(n=5) 1 0 0	(n=5) 0 1 1	(n=5) 0 0 0	(n=5) 0 0 0	(n=1) 0 0 0
Thyroid - dark, slight - pale, slight - small, slight	(n=10) 0 0 0	(n=10) 0 0 0	(n=10) 0 0 0	(n=9) 1 0 0	(n=8) 0 1 1	(n=10) 0 0 0	(n=8) 0 2 1	(n=1) 0 0 0
Thymus - dark, minimal - red focus - small, slight	(n=5) 0 0 0	(n=5) 1 0 0	(n=5) 0 1 0	(n=5) 0 1 0	(n=5) 0 0 0	(n=5) 0 0 0	(n=5) 0 0 1	(n=1) 0 0 0
Lymph node, mesenteric - red, slight	(n=5) 0	(n=5) 2	(n=5) 0	(n=5) 1	(n=5) 1	(n=5) 1	(n=5) 1	(n=1) 0
Liver - mottled, moderate - mottled, slight - pale, moderate - pale, slight	(n=7) 1 1 0 2	(n=5) 0 4 0 1	(n=7) 0 3 1 2	(n=7) 1 3 0 2	(n=5) 0 0 0 0	(n=5) 0 0 0 0	(n=5) 0 0 0 0	(n=1) 0 0 0 0
Stomach - raised focus, pale - red, slight - striation, red - thick, minimal - thick, slight	(n=5) 0 1 0 0 0	(n=5) 0 1 0 0 0	(n=5) 0 1 2 1 1	(n=5) 1 0 2 0 1	(n=5) 0 0 0 0 0	(n=5) 0 1 0 0 0	(n=5) 0 0 0 0 0	(n=1) 0 0 0 0 0
Colon - distension, slight	(n=5) 0	(n=5) 0	(n=5) 1	(n=5) 0	(n=5) 0	(n=5) 0	(n=5) 0	(n=1) 0
Lymph node, mandibular - red, moderate	(n=5) 0	(n=5) 1	(n=5) 0	(n=5) 0	(n=5) 0	(n=5) 0	(n=5) 0	(n=1) 0
Kidney - mottled, minimal - mottled, moderate - mottled, slight - pale, moderate - pale, slight - pelvic dilation, minimal - pelvic dilation, slight - red area	(n=5) 0 0 0 0 0 0 0 0	(n=5) 0 0 2 0 2 0 0 1	(n=8) 0 1 5 1 0 1 1 0	(n=7) 1 1 2 1 0 0 0 0	(n=5) 0 0 0 0 0 0 0 0	(n=5) 0 0 0 0 0 0 0 0	(n=5) 0 0 0 0 0 0 1 0	(n=5) 0 0 0 0 0 0 0 0
Testis - small, severe	(n=10) 0	(n=10) 1	(n=10) 0	(n=9) 0	n/a	n/a	n/a	n/a
Epididymis - small, marked	(n=10) 0	(n=10) 1	(n=10) 0	(n=9) 0	n/a	n/a	n/a	n/a
Skin - fur loss, moderate - fur loss, slight - sore, slight	(n=0) 0 0 0	(n=0) 0 0 0	(n=0) 0 0 0	(n=0) 0 0 0	(n=0) 0 0 0	(n=2) 1 0 1	(n=2) 0 2 1	(n=0) 0 0 0
Lung - pale focus	(n=5) 0	(n=5) 0	(n=5) 0	(n=5) 0	(n=5) 0	(n=5) 0	(n=5) 1	(n=1) 0
Tail - kinked	(n=0) 0	(n=0) 0	(n=0) 0	(n=0) 0	(n=2) 2	(n=0) 0	(n=0) 0	(n=0) 0
Peyer’s Patch - prominent, moderate	(n=5) 0	(n=5) 0	(n=5) 0	(n=5) 0	(n=5) 1	(n=5) 0	(n=5) 0	(n=1) 0
Mammary gland - thick, moderate	(n=10) 0	(n=10) 0	(n=10) 0	(n=9) 0	(n=8) 0	(n=10) 1	(n=8) 0	(n=1) 0

* P<0.05, ** P<0.01

# excluded from dataset

 

Table 7      Absolute and adjusted organ weights (F0)

 

Doses (mg/kg/day)	Control	100	300	500	Control	100	300	500
	MALE				FEMALE
BODY WEIGHT
Weight (g)	391.8	407.9	381.0	356.0*	249.8	253.8	252.2
TESTES
- Absolute weight (g)	3.561	3.534	3.724	3.781	n/a	n/a	n/a	n/a
- % body weight	0.912	0.878	0.982	1.068	n/a	n/a	n/a	n/a
EPIDIDYMIS
- Absolute weight (g)	1.413	1.320	1.431	1.346	n/a	n/a	n/a	n/a
- % body weight	0.362	0.327	0.377	0.381	n/a	n/a	n/a	n/a
SEMINAL VESICLE
- Absolute weight (g)	0.843	0.816	0.827	0.805	n/a	n/a	n/a	n/a
- % body weight	0.216	0.199	0.214	0.228	n/a	n/a	n/a	n/a
PROSTATE
- Absolute weight (g)	1.002	0.957	0.969	0.776***	n/a	n/a	n/a	n/a
- % body weight	0.256	0.236	0.254	0.219**	n/a	n/a	n/a	n/a
BRAIN
- Absolute weight (g)	2.071	2.069	2.015	2.026	1.883	1.946	1.961	nd
- % body weight	0.519	0.484	0.526	0.585*	0.767	0.733	0.768	nd
ADRENAL
- Absolute weight (g)	0.070	0.069	0.070	0.076	0.086	0.076	0.093	nd
- % body weight	0.018	0.016	0.018	0.022	0.035	0.029*	0.036	nd
SPLEEN
- Absolute weight (g)	0.605	0.651	0.554	0.526	0.577	0.504	0.491	nd
- % body weight	0.152	0.152	0.144	0.152	0.235	0.189**	0.191**	nd
THYMUS
- Absolute weight (g)	0.390	0.468	0.343	0.332	0.231	0.274	0.212	nd
- % body weight	0.098	0.109	0.090	0.096	0.093	0.104	0.082	nd
LIVER
- Absolute weight (g)	9.050	10.658**	11.517**	12.538**	9.184	8.927	10.809	nd
- % body weight	2.272	2.489	2.993*	3.594***	3.759	3.346	4.206	nd
HEART
- Absolute weight (g)	1.102	1.078	1.002	0.938***	0.859	0.798	0.849	nd
- % body weight	0.277	0.252	0.261	0.269	0.353	0.301	0.331	nd
KIDNEY
- Absolute weight (g)	2.257	2.473	2.717	2.426	1.841	1.747	1.864	nd
- % body weight	0.567	0.576	0.707*	0.699*	0.755	0.656	0.723	nd
PITUITARY
- Absolute weight (g)	0.010	0.009	0.010	0.009	0.012	0.012	0.013	nd
- % body weight	0.003	0.002	0.002	0.003	0.005	0.005	0.005	nd
THYROID/ PARA
- Absolute weight (g)	0.023	0.023	0.026	0.024	0.019	0.019	0.022	nd
- % body weight	0.006	0.006	0.007**	0.007*	0.008	0.007	0.009	nd
OVARY
- Absolute weight (g)	n/a	n/a	n/a	n/a	0.110	0.112	0.112	nd
- % body weight	n/a	n/a	n/a	n/a	0.044	0.044	0.044	nd
UTERUS
- Absolute weight (g)	n/a	n/a	n/a	n/a	0.501	0.457	0.496	nd
- % body weight	n/a	n/a	n/a	n/a	0.203	0.181	0.197	nd

* P<0.05, ** P<0.01, ***p<0.001

nd: not determined

Conclusions:

Test article-related findings observed in males following 100 mg/kg/day administration were generally confined to microscopic changes in the liver and thyroid, and a rat specific change in the kidneys. These findings were considered not to represent an adverse health effect in humans, and hence, the no observed adverse effect level (NOAEL) for male systemic toxicity was established as 100 mg/kg/day.

No test article-related changes were noted for maternal females or offspring following administration of 100 mg/kg/day. As such, the no observed effect level (NOEL) for maternal systemic toxicity, and the subsequent progeny, was established as 100 mg/kg/day.

Executive summary:

OECD 422 (2018) - In a combined repeat dose toxicity study with reproductive toxicity screening (OECD 422), Amines, C12-12-branched alkyl, docdecylbenzenesulfonates (1:1) was administered to 30 male and 30 female Crl:WI(Han) strain rats by oral gavage administration at dose levels of 100, 300 and 500 mg/kg/day for up to 6 weeks. In addition, 10 male and 10 female were used as control rats and were treated with the corn oil vehicle only.

 

A summary of adult responses to the test item are described below;

 

Males

 

One male administered 500 mg/kg/day was found dead on Study Day 16. No adverse clinical observations were noted, and no specific cause of demise could be determined macroscopically or microscopically. The cause of death in relation to the test article could not be excluded.

 

Urogenital staining and wet abdominal fur was recorded occasionally for males administered 300 or 500 mg/kg/day. During the FOB assessment, salivation; piloerection; changes in body posture; and a dose‑related increase in ataxia, high stepping, walking on toes, and slow deliberate movements were noted. Additionally, increased incidences of rears were noted for males administered 300 or 500 mg/kg/day, compared with controls. These observations were considered an adverse test article-related effect.

 

The most marked reduction in body weight gain was noted during the pre‑pairing phase, with a statistically significant reduction noted for males administered 500 mg/kg/day. However, reduction in body weight gain continued to be noted, and the mean body weight gain after 6 weeks of dosing was statically significantly reduced for males administered 300 or 500 mg/kg/day, compared with controls. This reduction in body weight gain was not associated with a reduction in food consumption.

 

Clinical pathology parameters affected include increased glucose, cholesterol, calcium, inorganic phosphate, and increased urine output for animals administered 300 or 500 mg/kg/day. Mean water consumption was also increased, but as water consumption values were only collected for the first 2 weeks of study, this increase suggested test article-related kidney damage. This was also supported by microscopic observations noted; nephropathy and hyaline droplet accumulation in the renal tubules. These observations may indicate enhanced or accelerated development of glomerulosclerosis.

 

The epidermal hyperplasia and/or hyperkeratosis in the nonglandular stomach were considered consistent with a chronic irritation-type reaction.

 

Hepatocyte hypertrophy in the liver and the associated increase in TSH and thyroid follicular cell hypertrophy is commonly observed as an adaptive response associated with the metabolism of xenobiotics or their metabolites and is considered rat specific.

 

Females

 

In general, females were less affected than males. No test article-related effects on clinical or postdose observations were noted, although lower body weight gain and food consumption was noted during late gestation following 500 mg/kg/day administration.

 

Some markers of toxicity were observed. The FOB observations included piloerection, salivation, and paw flicking (fore paws), which increased in a dose‑dependent manner. A decreased number of rears was also noted during the lactation phase for females 300 mg/kg/day, compared with controls. Mean water consumption was increased for animals administered 300 or 500 mg/kg/day, at 38 or 80 % greater than the controls, respectively. Cholesterol and glucose levels were also elevated in females administered 300 mg/kg/day. However, the macroscopic and/or microscopic observations noted in the kidney of males were not apparent in females.

 

Microscopic observations included epidermal hyperplasia and/or hyperkeratosis in the non-glandular stomach, which were considered consistent with a chronic irritation-type reaction.

 

Hepatocyte hypertrophy in the liver and the associated increase in TSH and thyroid follicular cell hypertrophy were less pronounced in females than males; nevertheless, it is commonly observed as an adaptive response associated with the metabolism of xenobiotics or their metabolites and is considered rat specific.

 

Endpoint Discussion

 

Test article-related findings observed in males following 100 mg/kg/day administration were generally confined to microscopic changes in the liver and thyroid, and a rat specific change in the kidneys. These findings were considered not to represent an adverse health effect in humans, and hence, the no observed adverse effect level (NOAEL) for male systemic toxicity was established as 100 mg/kg/day.

 

No test article-related changes were noted for maternal female administration of 100 mg/kg/day. As such, the no observed effect level (NOEL) for maternal systemic toxicity, and the subsequent progeny, was established as 100 mg/kg/day.

 

This combined toxicity study with reproduction screening in the rat is acceptable and satisfies the guideline requirements for an OECD 422 in the rat.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

28 April 2017 - 21 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with international guidelines and in accordance with GLP. The purpose of this study was the assessment of systemic toxic potential of the test item in a 14 day dietary study in the Crl:CD(SD) rat, to select suitable dose levels for a subsequent combined repeated dose toxicity study with the reproductive/developmental toxicity screening study (OECD TG 422).

Principles of method if other than guideline:

The purpose of this study was the assessment of systemic toxic potential of the test item in a 14 day oral gavage study in the Crl:WI(Han) rat, to select suitable dose levels for a subsequent combined repeated dose toxicity study with the reproductive/developmental toxicity screening study (OECD TG 422).

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: Homogenous suspension formed in corn oil.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Suspended in corn oil prior to administration
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: Not reported
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Applied as a liquid

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) N/A

OTHER SPECIFICS: N/A

Species:

rat

Strain:

other: CRL:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Margate, UK
- Females (if applicable) nulliparous and non-pregnant: Not reported
- Age at study initiation: 10-12 weeks
- Weight at study initiation: Males: 285.8 - 368.4 g; Females: 182.2 - 236.9 g
- Fasting period before study: Not reported
- Housing: Animals were housed in cages that conform to the Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes (Home Office, 2014). Animals were housed in groups of five by sex. Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips (Datesand Ltd, Manchester, United Kingdom)
- Diet (e.g. ad libitum): Animals had ad libitum access to 5LF2 EU Rodent Diet (International Product Supplies Ltd., London, United Kingdom). Each batch of diet was analyzed for specific constituents and contaminants. No contaminants were present in the diet at levels that may have interfered with achieving the objective of the study
- Water (e.g. ad libitum): Main supply water was provided ad libitum via water bottles. The water is periodically analyzed for specific contaminants. No contaminants were present in the water at levels that may have interfered with achieving the objective of the study.
- Acclimation period: 14-22 days

DETAILS OF FOOD AND WATER QUALITY: Asdescribed above.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 30-70 % RH
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: From: 23 May 2017 To: 13 June 2017

Route of administration:

oral: gavage

Details on route of administration:

Test article formulations (excluding the control group) were stirred upon arrival to the animal room from at least 30 minutes prior to, and continuously throughout, dosing.

Formulations were dosed within 3 hours of preparation.

The test article was administered orally by gavage. Animals were dosed once daily for up to 14 consecutive days, excluding the day of necropsy. A dose volume of 10 mL/kg was used. Dose volumes were based on the most recent individual body weight for each animal.

Vehicle:

corn oil

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:

Test article formulations (excluding the control group) were stirred upon arrival to the animal room from at least 30 minutes prior to, and continuously throughout, dosing. Formulations were dosed within 3 hours of preparation.

Method of preparation not reported in range-finder.

- DIET PREPARATION
- Rate of preparation of diet (frequency): N/A
- Mixing appropriate amounts with (Type of food): N/A
- Storage temperature of food: N/A

- VEHICLE
- Justification for use and choice of vehicle (if other than water): Test item formed homogenous formulation in corn oil. Guideline recommended vehicle.
- Concentration in vehicle: Not reported
- Amount of vehicle (if gavage): 10 mL/kg body weight
- Lot/batch no. (if required): Batch numbers MKBP7039V and MKBZ9899V
- Purity: Not reported, assumed 100 %

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

N/A

Duration of treatment / exposure:

14 days

Frequency of treatment:

Daily

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

750 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The initial dose level was based on the findings from an acute toxicity study in the rat, where a single dose of 2000 mg/kg was well tolerated (Study 8361291).
- Rationale for animal assignment (if not random): N/A
- Rationale for selecting satellite groups: N/A
- Post-exposure recovery period in satellite groups: N/A
- Section schedule rationale (if not random): N/A

Positive control:

N/A

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weely

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded once weekly during the predose phase; on Days 1 and 4 (all animals) and 8, 11, and 14 (Control, 500 and 750 mg/kg/day groups only) of the dosing phase; and before each necropsy

FOOD CONSUMPTION
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: No

Other examinations:

N/A

Statistics:

Statistical analyses were not performed.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Among surviving animals, stained skin, wet fur, salivation, and thinning fur were observed. Minimal salivation was observed in one male administered 500 mg/kg/day, one male administered 750 mg/kg/day, and one female administered 750 mg/kg/day. Yellow or orange staining of skin (abdomen, legs, nose, and urogenital area), wet fur around the urogenital area and fore legs were observed for animals administered 500 or 750 mg/kg/day, and thinning fur around the forelegs and urogenital area was observed for some animals administered 750 mg/kg/day.

Postdose observations included mouth rubbing, paddling, subdued/sluggish behavior, semi-closed eyes, raised hair, and salivation for animals administered ≥500 mg/kg/day, and reduced activity for animals administered ≥750 mg/kg/day. Mouth rubbing was also observed for controls; however, the duration and frequency of mouth rubbing was higher for test article-treated animals, compared with controls. Mouth rubbing, salivation, paddling, and raised hair were noted immediately upon return to the cage and up to 4 hours postdose. Subdued/sluggish behavior and reduced activity were observed within 30 minutes to 1 hour postdose. At the end of the day, raised hair, salivation, and mouth rubbing continued to be observed for animals administered ≥750 mg/kg/day; sluggish behavior was noted for one female administered 500 mg/kg/day; and reduced activity and sluggish behavior were observed for animals administered 1000 mg/kg/day. On the following morning, the only postdose observations were raised hair and sluggish activity for animals administered 1000 mg/kg/day.

Mortality:

mortality observed, treatment-related

Description (incidence):

Test article-related mortality was observed for animals administered 1000 mg/kg/day. Two females administered 1000 mg/kg/day were sacrificed in a moribund condition on Day 3 or 6, respectively. Prior to death, one animal was noted as hunched and subdued/sluggish; had loose feces, semi-closed eyes, and brown or yellow stained skin (urogenital area, anus, muzzle, nose, and front paws); and lost 31.7 g of body weight (Days 1 to 3). The second animal was moderately thin; had vocalization, raised hair, yellow stained skin, and wet fur (abdomen, urogenital area); and lost 22.2 g body weight (Days 1 to 6). All remaining animals administered 1000 mg/kg/day, were sacrificed on Day 7 of the dosing phase due to continued body weight losses and clinical observations. Clinical observations for these animals included one or more of the following: hunched, yellow stained skin (urogenital area, inguinal region, abdomen), and wet fur (urogenital area).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Test article-related body weight losses were observed for the interval of Days 1 to 4 for males administered ≥500 mg/kg/day. For males administered 1000 mg/kg/day, body weight losses continued to be observed for individual animals, until their sacrifice on Day 7. Lower mean body weight gains continued to be observed during the next two intervals (Days 4 to 8 and 8 to 11) for males administered 500 mg/kg/day, and for all remaining intervals of the dosing phase for males administered 750 mg/kg/day. Compared with controls, a higher mean body weight gain was noted for the last dosing phase interval (Days 11 to 14) for males administered 500 mg/kg/day. Compared with controls, overall body weight gain was 60% and ~100% lower, and Day 14 mean body weight was 6% and 16% lower, for males administered 500 or 750 mg/kg/day, respectively.

Test article-related body weight losses were observed for the interval of Days 1 to 4 for females administered ≥500 mg/kg/day. For all subsequent intervals during the dosing phase, mean body weight gain for females administered 500 or 750 mg/kg/day was generally comparable with that of controls therefore indicating the body weight effect was reversed. For females administered 1000 mg/kg/day, body weight losses continued to be observed for individual animals, until their sacrifice on Day 7.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Compared with controls, dose-dependent decreases in mean food consumption were noted during all dosing phase intervals (Days 1 to 4, 4 to 8, 8 to 11, and 11 to 14) for males administered ≥500 mg/kg/day. Decreased food consumption for these animals correlated with body weight loss or decreased body weight gain.

Compared with controls, dose-dependent decreases in mean food consumption were noted for the interval of Days 1 to 4 for females administered ≥500 mg/kg/day. For all subsequent intervals during the dosing phase, mean food consumption for females administered 500 or 750 mg/kg/day was comparable with that of controls. For the interval of Days 4 to 6, females administered 1000 mg/kg/day continued to have low food consumption. Decreased food consumption correlated with body weight loss or decreased body weight gain.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Compared with controls, mean water consumption was generally higher during the dosing phase for animals administered ≥500 mg/kg/day, except on Days 1 to 2 and
2 to 3, when mean water consumption was minimally lower for animals administered 1000 mg/kg/day. This decrease correlated with general debilitation noted for these animals.

Ophthalmological findings:

not examined

Description (incidence and severity):

N/A

Haematological findings:

not examined

Description (incidence and severity):

N/A

Clinical biochemistry findings:

not examined

Description (incidence and severity):

N/A

Urinalysis findings:

not examined

Description (incidence and severity):

N/A

Behaviour (functional findings):

not examined

Description (incidence and severity):

N/A

Immunological findings:

not examined

Description (incidence and severity):

N/A

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Group mean unadjusted epididymis (-5 and -12%) and testes (-4 and -10%) weights were lower for males administered 500 or 750 mg/kg/day, compared with concurrent controls. However, group mean body weight ratios for these organs were higher than for controls; therefore, these decreased unadjusted organ weights were considered to be related to body weight losses and not directly test article related.

Description (incidence and severity):

Macroscopic observations were recorded in the stomach, liver, kidney, mesenteric lymph nodes, lungs, adrenal, skin, uterus, and testes. Liver findings were observed at all dose levels, and kidney and stomach findings were observed in animals administered ≥750 mg/kg/day.

Findings in the stomach included distension, dark or thick mucosa, irregular surface, pale appearance, or raised focus/area for two females administered 1000 mg/kg/day and three females administered 750 mg/kg/day.

Findings in the kidney included pale, mottled appearance, or pelvic dilatation in three males administered 1000 mg/kg/day and two males administered 750 mg/kg/day.

Liver findings included large, mottled, or pale appearance in two males administered 1000 mg/kg/day (Animals R0301 and R0304), two males administered 750 mg/kg/day, one female administered 750 mg/kg/day, and one female administered 1000 mg/kg/day. Incidence of findings in the kidney, liver, and stomach exhibited dose-response.

Based on the incidence, findings in the stomach, liver, and kidney were considered potentially test article related. Findings in other tissues were observed in a single animal or lacked a dose-response; therefore, a relationship to the test article could not be ruled out.

Neuropathological findings:

not examined

Description (incidence and severity):

N/A

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

N/A

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

N/A

Other effects:

not examined

Description (incidence and severity):

N/A

Table 3      Mean body weights and body weight gains/ losses

Sex / Day	Bodyweight (g)
	Control	500 ppm	750 ppm	1000 ppm
MALES
Pre-dose Day 1	298.5	303.5	279.6	303.5
Pre-dose Day 8	-	-	307.6	323.7
Day 1	333.4	335.4	312.6	328.2
Day 4 (change from Day 1-4)	339.7 (6.3)	332.4 (-3.0)	306.6 (-6.0)	313.6 (-14.6)
Day 8 (change from Day 4-8)	354.5 (14.8)	341.2 (8.8)	307.9 (1.2)	- (-)
Day 11 (change from Day 8-11)	363.4 (8.9)	341.2 (0.1)	314.4 (6.5)	- (-)
Day 14 (change from Day 11-14)	370.9 (7.5)	350.4 (9.2)	312.5 (-1.9)	- (-)
Change from Day 1-14	37.5	15.0	-0.1	-
FEMALES
Pre-dose Day 1	199.2	206.6	188.3	205.3
Pre-dose Day 8	-	-	201.2	215.3
Day 1	205.3	211.6	200.2	218.0
Day 4 (change from Day 1-4)	205.9 (0.6)	203.8 (-7.8)	193.7 (-6.5)	199.6 (-14.0)
Day 8 (change from Day 4-8)	212.4 (6.5)	212.4 (8.6)	202.9 (9.2)	- (-)
Day 11 (change from Day 8-11)	214.4 (2.0)	213.0 (0.6)	205.1 (2.3)	- (2.3)
Day 14 (change from Day 11-14)	209.9 (-4.5)	214.1 (1.1)	210.2 (5.0)	- (-)
Change from Day 1-14	4.6	2.4	10.0	-

 

Table 4      Unadjusted organ weights (relative to body weight %)

Doses (ppm)	Control	7500	11000	3000	Control	7500	11000	3000
	Males				Females
Number of animals/group	5	5	5	0	0	0	0	0
BODY WEIGHT (termination weight for toxicity males)
Weight (g)	358.4	333.9	297.4	-	-	-	-	-
EPIDIDMYSIS (termination weight for toxicity males)
Weight (g)	1.279	1.214	1.128	-	-	-	-	-
% body weight	0.3583	0.3639	0.3789	-
TESTIS (termination weight for toxicity males)
Weight (g)	3.594	3.445	3.252	-	-	-	-	-
% body weight	1.0044	1.0370	1.0989	-	-	-	-	-

Conclusions:

In a 14-day OECD 422 dose range-finding study, daily oral gavage administration of 500, 750 or 1000 mg/kg/day Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1) to male and female rats was not tolerated following 750 or 1000 mg/kg/day administration. Based on the findings of this 14 day repeated dose study, dose levels of 500, 300 and 100 mg/kg/day were recommended for the following OECD 422 study.

Executive summary:

OECD 422 Range Finder (2018) - The purpose of this test was to assess the systemic toxicity potential of Amines, C12-14-branched alkyl, dodecylbenzenesulfonates (1:1) following repeated exposure to 20 male and 20 female Crl:WI(Han) strain rats by dietary administration at dose levels of 500, 750 and 1000 ppm for up to 14 days. The findings would be used to select suitable dose levels for a subsequent combined repeat dose toxicity study with reproductive/ developmental toxicity screening study (OECD 422).

 

A summary of adult responses to the test item are described below;

 

Mortality- Two females administered 1000 mg/kg/day were sacrificed in a moribund condition on Day 3 or 6, respectively, and all remaining animals administered 1000 mg/kg/day were sacrificed on Day 7 of the dosing phase due to adverse clinical observations and body weight loss.

 

Clinical signs- Generalised clinical observations which included stained skin, wet fur, salivation, thinning fur, subdued/sluggish behavior, salivation, paddling, raised hair and mouth rubbing were observed in the 500 and 750 mg/kg/day test groups.  Additionally, in the 750 mg/kg/day group reduced activity was observed.

A few postdose observations continued to be observed at the end of the day for animals administered 750 mg/kg/day, whereas postdose observations were generally resolved by the end of each day of dosing for animals administered
 500 mg/kg/day.

 

Bodyweight and food consumption- Administration of 500 or 750 mg/kg/day resulted in initial body weight losses, with correlating decreased food consumption. Decreased body weight gain and food consumption continued to be observed until the end of the dosing phase for males administered 750 mg/kg/day; whereas decreased body weight gain was reversed by the end of the dosing phase for animals administered 500 mg/kg/day and decreased food consumption was reversed by the end of the dosing phase for females administered 500 mg/kg/day.

 

Necropsy- Macroscopic findings were observed in the stomach and kidney for both sexes administered ≥750 mg/kg/day and in the liver for both sexes administered
 ≥500 mg/kg/day.

 

Organ weights- Compared with concurrent controls, group mean unadjusted epididymis (-5 and -12%) and testes (-4 and -10%) weights were lower for males administered 500 or 750 mg/kg/day, which were related to body weight losses and not directly test article related.

 

Based on these findings, a dose level of 750 mg/kg/day may not be tolerated for a longer duration of dosing due to findings of consistently decreased body weight gain and food consumption, in conjunction with the clinical observations of subdued behavior and reduced activity.

 

Dose levels of 500, 300 and 100 mg/kg/day are recommended for the following OECD 422 study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The endpoint is concluded based on a single key study with a Klimish rating of 1.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In accordance with Annex VIII, Section 8.6.1, Column 2 and supporting ECHA Guidance, concerning repeated dose toxicity testing, the oral route is the default one because it is assumed to maximise systemic availability (internal dose). There was no evidence available to support the use of an alternative route for this study.

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In accordance with Annex VIII, Section 8.6.1, Column 2 and supporting ECHA Guidance, concerning repeated dose toxicity testing, the oral route is the default one because it is assumed to maximise systemic availability (internal dose). There was no evidence available to support the use of an alternative route for this study.

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In accordance with Annex VIII, Section 8.6.1, Column 2 and supporting ECHA Guidance, concerning repeated dose toxicity testing, the oral route is the default one because it is assumed to maximise systemic availability (internal dose). There was no evidence available to support the use of an alternative route for this study.

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

During the treatment period, the main adverse effects observed at mid and high dose groups were local effects of the test item resulting into epithelial hyperplasia and orthokeratotic hyperkeratosis recorded in the stomach, which correlate with the increased mean water consumption .  These effects are considered relevant for humans.

Additional information

OECD 422 (2018) - In a combined repeat dose toxicity study with reproductive toxicity screening (OECD 422), Amines, C12-12-branched alkyl, docdecylbenzenesulfonates (1:1) was administered to 30 male and 30 female Crl:WI(Han) strain rats by oral gavage administration at dose levels of 100, 300 and 500 mg/kg/day for up to 6 weeks. In addition, 10 male and 10 female were used as control rats and were treated with the corn oil vehicle only.

 

A summary of adult responses to the test item are described below;

 

Males

 

One male administered 500 mg/kg/day was found dead on Study Day 16. No adverse clinical observations were noted, and no specific cause of demise could be determined macroscopically or microscopically. The cause of death in relation to the test article could not be excluded.

 

Urogenital staining and wet abdominal fur was recorded occasionally for males administered 300 or 500 mg/kg/day. During the FOB assessment, salivation; piloerection; changes in body posture; and a dose‑related increase in ataxia, high stepping, walking on toes, and slow deliberate movements were noted. Additionally, increased incidences of rears were noted for males administered 300 or 500 mg/kg/day, compared with controls. These observations were considered an adverse test article-related effect.

 

The most marked reduction in body weight gain was noted during the pre‑pairing phase, with a statistically significant reduction noted for males administered 500 mg/kg/day. However, reduction in body weight gain continued to be noted, and the mean body weight gain after 6 weeks of dosing was statically significantly reduced for males administered 300 or 500 mg/kg/day, compared with controls. This reduction in body weight gain was not associated with a reduction in food consumption.

 

Clinical pathology parameters affected include increased glucose, cholesterol, calcium, inorganic phosphate, and increased urine output for animals administered 300 or 500 mg/kg/day. Mean water consumption was also increased, but as water consumption values were only collected for the first 2 weeks of study, this increase suggested test article-related kidney damage. This was also supported by microscopic observations noted; nephropathy and hyaline droplet accumulation in the renal tubules. These observations may indicate enhanced or accelerated development of glomerulosclerosis.

 

The epidermal hyperplasia and/or hyperkeratosis in the nonglandular stomach were considered consistent with a chronic irritation-type reaction.

 

Hepatocyte hypertrophy in the liver and the associated increase in TSH and thyroid follicular cell hypertrophy is commonly observed as an adaptive response associated with the metabolism of xenobiotics or their metabolites and is considered rat specific.

 

Females

 

In general, females were less affected than males. No test article-related effects on clinical or postdose observations were noted, although lower body weight gain and food consumption was noted during late gestation following 500 mg/kg/day administration.

 

Some markers of toxicity were observed. The FOB observations included piloerection, salivation, and paw flicking (fore paws), which increased in a dose‑dependent manner. A decreased number of rears was also noted during the lactation phase for females 300 mg/kg/day, compared with controls. Mean water consumption was increased for animals administered 300 or 500 mg/kg/day, at 38 or 80 % greater than the controls, respectively. Cholesterol and glucose levels were also elevated in females administered 300 mg/kg/day. However, the macroscopic and/or microscopic observations noted in the kidney of males were not apparent in females. Eight females administered 500 mg/kg bw/day and one control and 300 mg/kg bw/day female had total litter losses, resulting in their early termination.

 

Microscopic observations included epidermal hyperplasia and/or hyperkeratosis in the non-glandular stomach, which were considered consistent with a chronic irritation-type reaction.

 

Hepatocyte hypertrophy in the liver and the associated increase in TSH and thyroid follicular cell hypertrophy were less pronounced in females than males; nevertheless, it is commonly observed as an adaptive response associated with the metabolism of xenobiotics or their metabolites and is considered rat specific.

 

Endpoint Discussion

 

Test article-related findings observed in males following 100 mg/kg/day administration were generally confined to microscopic changes in the liver and thyroid, and a rat specific change in the kidneys. These findings were considered not to represent an adverse health effect in humans, and hence, the no observed adverse effect level (NOAEL) for male systemic toxicity was established as 100 mg/kg/day.

 

No test article-related changes were noted for maternal female administration of 100 mg/kg/day. As such, the no observed effect level (NOEL) for maternal systemic toxicity, and the subsequent progeny, was established as 100 mg/kg/day.

 

This combined toxicity study with reproduction screening in the rat is acceptable and satisfies the guideline requirements for an OECD 422 in the rat.

Justification for classification or non-classification

The substance does not meet the criteria for classification for specific target organ toxicity - repeated exposure (STOT-RE) in accordance with Regulation (EC) No 1272/2008 (CLP).