Reaction mass of 2-dodecylhexadecyl methacrylate and 2-Tetradecyloctadecyl methacrylate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

FROM 29 DEC 2020 to 28 JUL 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2-dodecylhexadecyl methacrylate and 2-Tetradecyloctadecyl methacrylate

EC Number:

946-978-0

IUPAC Name:

Reaction mass of 2-dodecylhexadecyl methacrylate and 2-Tetradecyloctadecyl methacrylate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier): Sponsor.
- Lot/batch number of test material: F1-0S002C
- Purity, including information on contaminants, isomers, etc.: 93.3 % w/w

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At ambient temperature (15 to 25°C) in the dark.

FORM AS APPLIED IN THE TEST
- Pale yellow liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: Males 71 to 77 days old; Females 84 to 97 days old.
- Weight at study initiation: Males 341 to 394 g; Females 254 to 314 g.
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: Males: Seven days before commencement of treatment. Females: 21 days before commencement of treatment

DETAILS OF FOOD AND WATER QUALITY:
The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 40-70%
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light: 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

VEHICLE
- Amount of vehicle (if gavage): 5 mL/kg

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Males: Two weeks before pairing up to necropsy after minimum of five weeks.
Females: Two weeks before pairing, then throughout pairing and gestation until Day 12 of lactation.
Animals of the F1 generation were not dosed.

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

low dose

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

mid dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

high dose

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cages were inspected daily for evidence of animal ill-health amongst the occupants. During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males: Before dosing on the day that treatment commenced (Week 0) and weekly thereafter.
On the day of necropsy.
F0 females: Before dosing on the day that treatment commenced (Week 0) and weekly before pairing. Days 0, 7, 14 and 20 after mating. Day 1, 4, 7, 11 and 13 of lactation. On the day of necropsy.

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: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes ( isoflurane)
- Animals fasted: Not specified
- How many animals: 5 lowest numbered surviving males per group. The first 5 lactating females with a surviving litter per group.
- Parameters examined:
Hematocrit (Hct)
Hemoglobin concentration (Hb)
Erythrocyte count (RBC)
Absolute reticulocyte count (Retic)
Mean cell hemoglobin (MCH)
Mean cell hemoglobin concentration (MCHC)
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Differential leucocyte count: Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC)
Platelet count (Plt)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Not specified
- How many animals: 5 lowest numbered surviving males per group. The first 5 lactating females with a surviving litter per group.
- Parameters examined:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Bile acids (BiAc)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb

SERUM HORMONES (Thyroid Hormone Analysis): Yes
- Time of blood sample collection: F0: at necropsy, F1 at day 4 and day 13 of age
- Animals fasted: Not specified
- How many animals:
All surviving F0 males and females.
At day 4 of age, offspring: up to two females per litter (where possible; male pups were reserved for nipple retention evaluation): one for T4 (serum), one for TSH (serum)
At day 13 of age, F1 offspring, two males and two females per litter (where possible) two for T4 (serum); where possible one male and one female, two for TSH (serum); where possible one male and one female

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: For males during Week 5, for females day 7-9 of lactation
- Dose groups that were examined: 5 lowest numbered surviving males in each group during Week 5 of treatment and on the first five lactating females in each group at Day 7-9 of lactation.
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see tables 1 and 2)

HISTOPATHOLOGY: Yes (see tables 1 and 2)

Statistics:

Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant, are presented on the relevant tables. For some parameters, including estrous cycles before treatment, estrous cycles during treatment, pre-coital interval, mating performance, gestation length and index and stage of estrous cycle at termination, the similarity of the data was such that analyses were not considered to be necessary.

All statistical analyses were carried out separately for males and females. Data relating to food consumption (except during gestation and lactation) were analyzed on a cage basis. For all other adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level. The stastical methods used are given below

Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-tests.

Comparison of all groups using Chi-square test

For grip strength, motor activity, body weight, food consumption, corpora lutea, implantations, pre/post implantation loss, live young, sex ratio - percentage male, placental, litter and fetal weights, ano-genital distance, organ weight and clinical pathology data:
Treated groups compared with Control using Dunnett’s test
Treated groups compared with Control using Shirley’s test
Treated groups compared with Control using Williams’ test
Treated groups compared with Control using Fisher’s exact test

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were observed for either sex pre- or post-pairing or during gestation and lactation for females. One male receiving 1000 mg/kg bw/day was observed with dry rales following dose administration on Day 20. There were no signs seen in females post-dose administration throughout the study.

All other signs observed were considered incidental and not related to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control female (No. 127) was found dead on Day 7 of gestation during the morning cage side checks. On Day 6 of gestation this female was showing signs of irregular breathing, piloerection, hunched posture and chromodacryorrhea. Macroscopic findings included yellow abnormal contents in the duodenum and jejunum, abnormal contents in the thoracic cavity (pale caseous material, clear fluid, food material and clear oily material), pale fibrous adhesions affecting multiple organs and a perforated oesophagus. The female was pregnant with 16 implantations. Histopathological evaluation revealed inflammation and presence of foreign material in the thoracic cavity and inflammation of the heart. The thoracic cavity and heart lesions are indicative of dosing trauma, which was considered responsible for the animal’s death.

One female that received 1000 mg/kg bwday (No. 114) was euthanized for welfare reasons on Day 1 of lactation. The animal had decreased activity, irregular breathing, piloerection, vaginal bleeding, dull eyes, and whole-body pallor. Macroscopic findings included dark fluid present in the cecum, jejunum, rectum, thoracic cavity and vagina, an enlarged lumbar lymph node, edematous thymus and a pale area on the left ventricle of the heart and many pale organs. Histopathological findings included inflammation of the heart, extramedullary hemopoiesis of the spleen and liver, edematous thymus and increased generalized cellularity of the lumbar lymph node. The heart and thymus lesions were indicative of dosing trauma, despite no findings in the esophagus, which was considered to be responsible for the terminal condition of the animal.

One female that received 100 mg/kg bw/day (No. 109) was dispatched to necropsy for welfare reasons on Day 2 of lactation due to general poor clinical condition and a swollen area on the upper ventral surface. This female had 17 offspring (8 male and 9 female) and on Day 2 of lactation two male offspring were found dead, one female offspring was missing, and the remaining pups were abnormally cold to touch with little or no milk present in their stomachs. Macroscopic findings at necropsy revealed hair loss of the fore and hind limbs. No histopathological evaluation was done for this animal in accordance with the study plan. Given the single incidence in the low-dose group, this premature death was not considered to be related to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Effect on body weight performance comprising low body weight gain prior to pairing (Weeks 1-2) was observed for males receiving 300 or 1000 mg/kg bw/day, which was not associated with reduced food intake. Body weight gains were slightly high for females receiving 1000 mg/kg bw/day from the second week of gestation onwards, corresponding with higher food intake, and from Days 7 to 11 of lactation with no associated increase in food consumption. Since there was no effect of treatment on the clinical condition of the F0 males and females, no effect on offspring survival or development and in the absence of any dose-relationship the extent of these body weight differences was of no toxicological importance

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The hematological examination of peripheral blood performed in males in Week 5, when compared with controls, revealed low reticulocyte and high basophil counts at all dose levels although no dose relationship was observed. Mean cell hemoglobin content and concentration was also high at 300 or 1000 mg/kg bw/day, but again, no dose relationship was apparent. These differences from control were considered not treatment related due to the lack of any dose relationship.

The hematological examination of peripheral blood performed in females on Day 13 of lactation, when compared with controls, revealed high hemoglobin concentrations at 1000 mg/kg bw/day. Total leucocyte counts and total lymphocyte count were high for males that received 100 or 300 mg/kg/day. However, these are considered to be incidental and not an effect of treatment as values at 1000 mg/kg/day were similar to controls. All other differences from controls were minor and were therefore attributed to normal biological variation.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The biochemical examination of the blood plasma performed in males after 5 weeks of treatment, when compared with controls, revealed low glucose and phosphorus concentrations at 1000 mg/kg bw/day. The biochemical examination of the blood plasma performed in females on Day 13 of lactation, when compared with controls, revealed high chloride and phosphorus concentrations at 1000 mg/kg bw/day. All other differences from controls were minor and were therefore attributed to normal biological variation.

Endocrine findings:

effects observed, treatment-related

Description (incidence and severity):

The serum T4 concentrations show a dose dependent increase in F0 males with concentrations, relative to control, increased by +15% (at 100 mg/kg bw/day), +20% (at 300 mg/kg bw/day) and +24% (at 1000 mg/kg bw/day). Statistical significance was obtained for rats tested at 300 mg/kg bw/day and rats tested at 1000 mg/kg bw/day only. However, there were no microscopic and macroscopic findings in the thyroid or liver, no organ weight changes and no increased body weight gain in the treated F0 males (for further details see 'Any information on results' section. ). There were no other statistically significant differences observed between the treated groups the control.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related organ weight changes were seen. Several intergroup differences between controls and treated animals were observed. These included lower absolute and relative heart weights in treated males, which attained statistical significance (relative weight) in males administered 300 or 1000 mg/kg/day. Other variations in weights included lower/higher absolute or relative weights of the thymus of treated females, and spleen and Cowper’s glands of treated males.

Gross pathological findings:

no effects observed

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related finding was observed after at least 4 weeks of oral gavage administration of the test substance o Sprague Dawley rats.

All microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was as expected for Sprague Dawley rats of this age. Therefore, they were considered not test item-related.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

A qualitative evaluation of spermatogenesis was performed. The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: absence of adverse treatment-related effects observed

2

Dose descriptor:

LOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

serum/plasma hormone analyses

Remarks on result:

other: Dose-dependent increase in T4 concentration in F0 males without microscopic findings in thyroid and liver, without changes in organ weights and without increase in body weight gain in treated F0 males.

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Table 3: Mean serum T4 concentration (pg/mL) in offsprings and F0 males

Group	Treatment	Dose   (mg/kg/day)	Parameters	F1 offspring on Day 13 of age  (Male)	F1 offspring on Day 13 of age  (Female)
1	Control (Vehicle)^a	0	Mean	59300	51700
			SD	11000	16800
			CV%	18.5	32.5
			N	9	9
2	Reaction mass of 2-Propenoic acid, 2-methyl-, 2- dodecylhexadecyl ester and 2-Propenoic acid, 2-methyl-, 2-tetradecyloctadecyl ester	100	Mean	59500	62700
			SD	13500	26200
			CV%	22.7	41.8
			N	9	9
3		300	Mean	61800	61700
			SD	14200	13000
			CV%	23.0	21.1
			N	10	10
4		1000	Mean	58600	60400
			SD	8080	11900
			CV%	13.8	19.7
			N	9	9
Group	Treatment	Dose   (mg/kg/day)		Parameters	F0 Adult Terminal Male
1	Control (Vehicle)^a	0		Mean	49100
				SD	7990
				CV%	16.3
				N	10
2	Reaction mass of 2-Propenoic acid, 2-methyl-, 2- dodecylhexadecyl ester and 2-Propenoic acid, 2-methyl-, 2-tetradecyloctadecyl ester	100		Mean	56700
				SD	12300
				CV%	21.7
				N	10
3		300		Mean	58800*
				SD	7830
				CV%	13.3
				N	10
4		1000		Mean	60700*
				SD	11100
				CV%	18.3
				N	10

_{a – Corn oil}

_{* - Statistically significant difference observed (p value < 0.05) between treatment and control group (Group 1) using a Williams’ test (two tailed).}

Table 4: Serum concentrations of T4 in individual F0 adult male animals at termination after receiving the vehicle (corn oil) by daily oral gavage administration

Dose	Animal number Concentration (pg/mL)
Control	21	22	23	24	25	26	27	28	29	30
	47200	49300	57000	40200	57600	52000	44700	61200	45100	36200
100 mg/kg bw/day	1	10	2	3	4	5	6	7	8	9
	49400	62300	57100	59800	52200	86600^-	46600	53300	41200	58600
300 mg/kg bw/day	31	32	33	34	35	36	37	38	39	40
	51200	63100	50700	61500	57300	46500	63900	58600	61400	73500^-
1000 mg/kg bw/day	11	12	13	14	15	16	17	18	19	20
	51500	59300	58900	86000^-	51500	64100	65300^-	45700	58300	66800^-

^-individual outliners above the historical control data range

Table 5: Historical control data (HCD) for F0 males (presented as percentiles)

Study	Start Date	Animal	T4 Concentration (pg/mL)
1	22-Jul-20	11	39100
		12	44900
		13	42600
		14	51300
		15	54000
		16	45700
		17	46700
		18	41100
		19	37000
		20	59300
2	04-Sep-19	11	28500
		12	42800
		13	44400
		14	39100
		15	49500
		16	60900
		17	64700
		18	41900
		19	24200
		20	38700
3	12-Jul-19	1	34800
		2	49300
		3	36300
		4	62200
		5	59200
		6	48700
		7	42900
		8	43200
		9	53000
		10	52200
4	13-Mar-19	16	53700
		17	54100
		18	52000
		19	53100
		20	48600
5	21-Oct-18	1	49000
		2	40700
		3	53200
		4	31500
		6	44100
		7	54400
		8	59100
		9	54000
		10	54000
6	10-Oct-18	1	40300
		2	50400
		3	56300
		4	45900
		5	49200
		6	58700
		7	44800
		8	65000
		9	60100
		10	48500
7	18-Jun-18	16	37000
		17	43200
		18	39800
		19	38800
		20	60500
8	10-May-18	16	41000
		17	52800
		18	31700
		19	44000
		20	46600
		n	64
		0.010	26909.000
		0.050	32165.000
		0.500	47600.000
		0.950	60840.000
		0.990	64811.000
		mean	47410.938
		sd	8944.4869

 

 

 

 

 

Applicant's summary and conclusion

Conclusions:

Under the given conditions of this combined repeated dose toxicity study and reproductive/developmental toxicity screening study in male and female rats, the NOAEL was determined to be 1000 mg/kg bw/day for general systemic toxicity based on the absence of adverse treatment-related effects observed.

Executive summary:

In a Combined Repeated Dose Toxicity Study and Reproductive/Developmental Toxicity Screening Study (according to OECD TG 422) Reaction mass of 2-Propenoic acid, 2-methyl-, 2-dodecylhexadecyl ester and 2-Propenoic acid, 2-methyl-, 2-tetradecyloctadecyl ester was administered to 10 Sprague-Dawley rats/sex/dose by oral gavage at dose levels of 0, 100, 300 or 1000 mg/kg bw/day.

Treatment at dose levels up to 1000 mg/kg bw/day was well tolerated. There were no treatment-related premature deaths, no treatment-related signs were observed post-dose, no treatment-related changes in clinical condition of the F0 males or females or their offspring and no findings at macroscopic examination of the F0 adults or their offspring considered to be treatment related. Sensory reactivity, grip strength and motor activity investigations were unaffected by treatment. 

Effect on body weight performance comprising low body weight gain prior to pairing (Weeks 1-2) was observed for males receiving 300 or 1000 mg/kg bw/day, which was not associated with reduced food intake. Body weight gains were slightly high for females receiving 1000 mg/kg bw/day from the second week of gestation onwards, corresponding with higher food intake, and from Days 7 to 11 of lactation with no associated increase in food consumption.

Findings in the blood were the result of secondary consequences of the effects of treatment or attributable to stress. Such findings in males included the low reticulocyte counts at all dose levels and high mean cell hemoglobin content and concentrations at 300 or 1000 mg/kg/day and high hemoglobin concentrations in females given 1000 mg/kg/day. These changes were considered non-adverse. For animals receiving 1000 mg/kg bw/day there were no macroscopic or microscopic associations for the low glucose and phosphorus concentrations observed in males and high chloride and phosphorus concentrations observed in females. These changes were therefore considered non-adverse and of uncertain relationship to treatment. The serum T4 concentrations showed a dose-dependent increase in F0 males with concentrations, relative to control, increased by +15% (at 100 mg/kg bw/day), +20% (at 300 mg/kg bw/day) and +24% (at 1000 mg/kg bw/day). Statistical significance was obtained only for rats tested at 300 mg/kg bw/day and for rats tested at 1000 mg/kg bw/day. However, no microscopic or macroscopic observations of the thyroid or liver, no changes in organ weights and no increase in body weight gain were observed in treated F0 males. No other statistically significant differences were observed between the treated and control groups.

As already mentioned above, no test item-related finding was observed in male and female rats after at least 4 weeks of oral gavage administration of the test substance at histopathology examination. All microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), and/or their severity was as expected for Sprague Dawley rats of this age. Therefore, they were considered not test item-related.

A qualitative evaluation of spermatogenesis was performed. The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.

 The NOAEL was determined to be 1000 mg/kg bw/day for general systemic toxicity based on the absence of adverse treatment-related effects observed and a LOEL was set at 100 mg/kg bw/day based on the increase in T4 without histological, macroscopical or organ weight findings.