Reaction mass of 5,8,11,14-Tetraoxaoctadecane and 5,8,11,14,17-Pentaoxaheneicosane

Endpoint summary

Description of key information

The registration substance was investigated for its repeated dose toxicity according to the OECD Guideline 422.  Indications of hemolysis and metabolic adaptation were found.  No significant toxic effect was found up to the dose of 750 mg/kg bw.
For the assessment of the chronic toxicity of the registration substance, the read-across approach was introduced. Based on the knowledge on the mode of action/kinetic profile/chronic toxicity of the read-across supporting substances, the hazard profile of the registrations substance after prolonged exposure was predicted to be comparable to that found in the OECD 422 study. No classification is assigned. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-11-09 to 2013-05-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline 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

Principles of method if other than guideline:

Health Effects guidelines, OPPTS 870.3650, Combined repeated dose Toxicity study with the Reproduction/ developmental Toxicity
screening Test. EPA 712-C-00-368, July 2000.
Commission Regulation (EC) No. 440/2008, L 142, Annex Part B, May 30, 2008
‘OECD Series on principles of Good Laboratory Practice and compliance monitoring’ Document No 13 ENV/JM/MONO (2002) 9.

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: males: 9-10 weeks old, females: 8-9 weeks old.
Body weight at the allocation of the animals to the experimental groups: Females: 171 to 202 g, (mean: 186.50 g, ± 20%= 37.30 g)
Males: 237 to 285 g, (mean: 264.21 g, ± 20%= 52.84 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3°C
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control,
microbiological controls at regular intervals)
- The animals were kept individually in IVC cages (except during the mating period when one female will be paired with one male),
type III H, polysulphone cages on Altromin saw fibre bedding
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made.
Before the first administration all animals used for the study were weighed and assigned to the experimental groups
with achieving a most homogenous variation in body weight throughout the groups of males and females.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The test item was weighed into a tared plastic vial on a precision balance and was suspended by adding the required volume of corn oil and
further vortexing it for 2-3 minutes. Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension
thoroughly before every dose administration. The test item formulation was prepared freshly on each administration day before the
administration procedure. The vehicle was used as control item.

The following doses were evaluated:
Control: 0 mg/kg bw
LD: 100 mg/kg bw
MD: 250 mg/kg bw
HD: 750 mg/kg bw

The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering.
Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
The doses were selected on the basis of data from a Dose Range Finding Study.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle
using the same dose volume.

Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 4 mL/kg body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each dosing concentration was analysed for nominal concentration and homogeneity of the test item in the vehicle at various intervals.
Stability analysis was performed on samples collected from the top and bottom dose levels.
Samples for the nominal concentration verification was taken in study week 1 (first week of pre mating period), study week 3 (first week of mating),
study week 5 (gestation) and study week 7 (gestation/lactation).
Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation in study week 1 and 5.
Samples for stability analysis was taken from top and bottom dose level in study week 1 at 0 hr and 6 hrs.
All concentration samples were stored frozen (approximately -20°C) until the analysis was performed.
The dose formulation analysis was performed at GLP-certified contract laboratory IBACON GmbH, Arheilger Weg 17, D-64380 Rossdorf,
Germany.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 54 days,
i.e. during 14 days of pre-mating and 14 days of mating in both males and females, during the gestation period and
up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

No. of animals per sex per dose:

96 animals (12 females and 12 males /group) were included in the study.

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
Once before the first exposure, and once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and on lactation days in 5 randomly selected females (only lactating females were evaluated) outside the home cage using a functional observational battery of tests.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups.

FOOD CONSUMPTION:
- Food consumption was measured weekly on the corresponding days of the body weight measurements after the beginning of the
dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: yes

HAEMATOLOGY: Yes
The haematological examinations (Haematocrit, haemoglobin, erythrocyte count, total and differential leucocyte count, platelet count and blood clotting time) were made in all males (Control and HD groups), six selected males (LD and MD groups) and six selected females from Control, LD, MD and
HD groups. Blood samples were taken from abdominal aorta as a part of the procedure for killing the animals.




CLINICAL CHEMISTRY: Yes
Clinical biochemistry determinations to investigate major toxic effects in tissues and, specifically, effects on kidney and liver were performed on blood samples obtained from all males (Control and HD groups), six selected males (LD and MD groups) and six selected females of each groups. Investigations of serum included sodium, potassium, glucose, total cholesterol, urea, creatinine, total protein and albumin, two enzymes indicative of hepatocellular effects (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase).


URINALYSIS: Yes
Urinalysis was performed on the samples collected from six randomly selected males at the terminal sacrifice.


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in the week before the first treatment and during the last week of the treatment
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The males were sacrificed after the completion of the mating period (after a dosing period of 28 days) except half of the males in control and HD
groups, which were further kept for 14 days recovery post 28 days dose administration and then scheduled for necropsy. Females were subjected to necropsy on the respective post-natal day 4. Males and females were sacrificed using an anesthesia (e.g. ketamine/xylazin). The pups were killed by decapitation on post-natal day 4.
Dead pups and pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.
Non-pregnant females (animal no. 54, 60 and 95) were sacrificed on study day 26 from the day of sperm-positive vaginal smear as an evidence of
mating.
All animals were subjected to a detailed gross necropsy, which includes careful examination of the external surface of the body, all orifices and the
cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

The reproductive organs of all adult animals were weighed. The wet weight of the organs (liver, kidneys, adrenals, testes, epididymides, prostate,
seminal vesicles and coagulating glands, ovaries, uterus with cervix, thymus, thyroid/parathyroid glands, spleen, brain, pituitary gland, heart))
of all males of control and high dose groups (including the animals subjected to recovery), six randomly selected males of low and mid dose groups and six randomly selected females from each group were recorded as soon as possible. Paired organs were weighed separately.


The following tissues (brain (cerebrum, cerebellum and pons), ovaries (females), spinal cord, uterus with cervix (females),
liver, vagina (females), kidneys, testes (males), adrenal glands, epididymides (males), stomach, prostate and seminal vesicles
with coagulating glands as a whole (males), small and large intestines (including Peyer´s patches), urinary bladder,
thymus, lymphnodes (mesentric and axillary), Thyroid, peripheral nerve (e.g. sciatic nerve) with skeletal muscle,
spleen, bone with bone marrow (sternum), lung and trachea pituitary gland, mammary glands, oesophagus, heart, gross lesions)
of the same selected animals from each group were preserved in 10% neutral buffered formalin except eyes, testes and epididymides
that were fixed in Modified Davidson’s Fixative for approximately 24 hours before they were transferred to 10% neutral buffered formalin.

HISTOPATHOLOGY: Yes

All organs and tissues listed were evaluated from all males of the control and high dose group assigned to main or recovery necropsy,
as well as from the following six randomly selected females from the control and high dose group (assigned to main necropsy):
Females Nos.: 51, 53, 55, 56, 58, 59, 85, 87, 89, 90, 92, 94.
In addition, liver, kidney, spleen and thyroid gland were also evaluated from the following six randomly selected males and females from the
low dose and medium dose groups: Males Nos.: 13, 15, 19, 21, 22, 24, 27, 28, 29, 32, 33, 36; Females Nos.: 63, 64, 66, 68, 71, 72, 73, 74, 75,
80, 81, 83.

Reproductive organs (ovary, uterus, cervix, vagina, testis, epididymis, prostate gland, seminal vesicle and coagulating gland) and
macroscopic changes were evaluated in all study animals.

For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of
additional hematoxylin-PAS (Periodic Acid Schiff) stained slides.

Histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory Propath UK Ltd.
(test site for tissue processing), Willow Court, Netherwood Road, Hereford HR2 6JU, England. Histopathological evaluation was
performed at the GLP-certified contract laboratory KALEIDIS – Consultancy in Histopathology (test site for histopathology),
6 rue du Gers, 68300 Saint-Louis, France. Blocking, embedding, cutting, H&E staining and scientific slide evaluation were
performed according to the corresponding SOP’s of the test sites.

Statistics:

For statistical analysis one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was carried out to reveal any differences between control- and test groups. The student-t test was used for the comparison of recovery control and recovery high dose groups.
These statistics were performed with GraphPad Prism V.5 software (p<0.05 was considered as statistical significant).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Table 1. Body weight development [g]
Observation Parameter		Dose
		Control	100 mg/kg	250 mg/kg	750 mg/kg
Males	Initial weight	299 ± 16 (n = 12)	298 ± 15 (n = 12)	296 ± 16 (n = 12)	298 ± 16 (n = 12)
	After premating phase	336 ± 23 (n = 12)	339 ± 20 (n = 12)	332 ± 26 (n = 12)	325 ± 19 (n = 12)
	After mating phase	361 ± 28 (n = 12)	361 ± 26 (n = 12)	356 ± 29 (n = 12)	350 ± 22 (n = 12)
	After recovery phase	380 ± 40 (n = 6)	-	-	359 ± 22 (n = 6)
Females	Initial weight	190 ± 10 (n = 10)	196± 8 (n = 12)	196± 8 (n = 12)	197± 8 (n = 11)
	After premating phase	203± 9 (n = 10)	211± 12 (n = 12)	209± 7 (n = 12)	206 ± 10 (n = 11)
	Gestation day 0	207± 8 (n = 10)	216± 13 (n = 12)	213± 9 (n = 12)	213 ± 10 (n = 11)
	Gestation day 20	316± 10 (n = 10)	320± 30 (n = 12)	317± 22 (n = 12)	312 ± 21 (n = 11)
	Lactation day 0	230± 12 (n = 10)	247± 20 (n = 12)	240± 18 (n = 12)	240 ± 13 (n = 11)
	Lactation day 4	242± 11 (n = 10)	259± 20 (n = 12)	248± 15 (n = 12)	250 ± 13 (n = 11)

 

Table 2. Hematology
Observation Parameter	At the treatment termination (one day after last treatment)				After 14 days of recovery
	Control	100 mg/kg	250 mg/kg	750 mg/kg	Control	750 mg/kg
Males
WBC [10^3/µL]	4.66 ± 0.46 (n = 6)	4.74 ± 0.73 (n = 6)	5.47 ± 0.65 (n = 5)	5.50 ± 1.33 (n = 5)	4.82 ± 0.85 (n = 6)	5.64 ± 1.25  (n = 6)
RBC [10^6/µL]	9.02 ± 0.25 (n = 6)	8.60 ± 0.34 (n = 6)	8.10 ± 0.49 * (n = 5)	8.00 ± 0.61 * (n = 5)	8.82 ± 0.24 (n = 6)	8.79 ± 0.42 (n = 6)
HGB [g/dL]	15.72 ± 0.66 (n = 6)	15.43 ± 0.27 (n = 6)	14.78 ± 0.49 (n = 5)	14.42 ± 1.03* (n = 5)	15.38 ± 0.26 (n = 6)	15.45 ± 0.55 (n = 6)
HCT [%]	45.87 ± 1.46 (n = 6)	44.68 ± 1.29 (n = 6)	42.60 ± 1.61 * (n = 5)	42.00 ± 2.82 * (n = 5)	45.42 ± 1.43 (n = 6)	46.35 ± 2.88 (n = 6)
MCV [fl]	50.83 ± 0.45 (n = 6)	52.03 ± 1.56 (n = 6)	52.68± 1.34 (n = 5)	52.56± 0.92 (n = 5)	51.48 ± 1.41 (n = 6)	52.75 ± 2.30 (n = 6)
MCH [pg]	17.40 ± 0.28 (n = 6)	17.98 ± 0.47 (n = 6)	18.26 ± 0.73 * (n = 5)	18.06 ± 0.23 (n = 5)	17.47 ± 0.59 (n = 6)	17.62 ± 0.43 (n = 6)
MCHC [g/dL]	34.25 ± 0.40 (n = 6)	34.60 ± 0.51 (n = 6)	34.66 ± 0.52 (n = 5)	34.32 ± 0.18 (n = 5)	33.92 ± 0.91 (n = 6)	33.40 ± 1.64 (n = 6)
PLT [10^3/µL]	857 ± 46 (n = 6)	802 ± 195 (n = 6)	871 ± 90 (n = 5)	961± 50 (n = 5)	856 ± 124 (n = 6)	854 ± 74 (n = 6)
PT [sec]	17.20 ± 0.56 (n = 6)	18.87 ± 7.67 (n = 6)	15.78 ± 0.89 (n = 5)	12.00 ± 0.44 (n = 5)	16.72 ± 0.60 (n = 6)	17.30 ± 0.72 (n = 6)
aPTT [sec]	19.62 ± 5.38 (n = 6)	26.57 ± 12.47 (n = 6)	32.52 ± 18.66 (n = 5)	51.52 ± 18.65 ** (n = 4)	26.47 ± 8.91 (n = 6)	39.58 ± 29.46 (n = 6)
Females
WBC [10^3/µL]	3.66 ± 0.30 (n = 6)	3.50 ± 1.19 (n = 6)	3.06 ± 0.45 (n = 6)	3.85 ± 0.60 (n = 6)	-	-
RBC [10^6/µL]	6.51 ± 0.41 (n = 6)	6.17 ± 0.55 (n = 6)	6.45 ± 0.37 (n = 6)	6.20 ± 0.35 (n = 6)	-	-
HGB [g/dL]	12.42 ± 0.83 (n = 6)	11.93 ± 0.65 (n = 6)	12.48 ± 0.66 (n = 6)	12.00 ± 0.56 (n = 6)	-	-
HCT [%]	37.12 ± 2.21 (n = 6)	35.67 ± 2.13 (n = 6)	36.70 ± 1.49 (n = 6)	36.47 ± 0.87 (n = 6)	-	-
MCV [fl]	57.08 ± 1.21 (n = 6)	58.02 ± 3.70 (n = 6)	56.98 ± 1.64 (n = 6)	58.95 ± 2.31 (n = 6)	-	-
MCH [pg]	19.08 ± 0.55 (n = 6)	19.40 ± 1.26 (n = 6)	19.38 ± 0.57 (n = 6)	19.37 ± 0.42 (n = 6)	-	-
MCHC [g/dL]	33.43 ± 0.49 (n = 6)	33.43 ± 0.66 (n = 6)	34.02 ± 0.91 (n = 6)	32.85 ± 0.92 (n = 6)	-	-
PLT [10^3/µL]	1269 ± 179 (n = 6)	1251± 149 (n = 6)	1239 ± 85 (n = 6)	1331 ± 186 (n = 6)	-	-
PT [sec]	16.76 ± 0.46 (n = 5)	16.62 ± 0.44 (n = 6)	16.56 ± 0.85 (n = 5)	17.05 ± 0.32 (n = 6)	-	-
aPTT [sec]	24.61 ± 12.35 (n = 5)	19.02 ± 4.81 (n = 5)	29.66 ± 14.11 (n = 5)	31.93 ± 10.37 (n = 6)	-	-

Table 3. Clinical Biochemistry
Observation Parameter	At the treatment termination (one day after last treatment)					After 14 days of recovery
	Control		100 mg/kg	250 mg/kg	750 mg/kg	Control	750 mg/kg
Males
ASAT [IU/L]	18.60± 5.17 (n = 6)		26.07± 6.50 (n = 6)	22.83± 6.19 (n = 6)	20.60± 5.51 (n = 5)	29.90 ± 5.32 (n = 6)	32.30 ± 23.19 (n = 6)
ALAT [IU/L]	18.58± 2.15 (n = 6)		24.52± 2.66* (n = 6)	23.40± 2.97 (n = 6)	24.62± 5.51* (n = 5)	19.27 ± 3.26 (n = 6)	35.02 ± 41.81 (n = 6)
ALP [IU/L]	126.17± 27.69 (n = 6)		194.50± 82.28 (n = 6)	161.17± 27.20 (n = 6)	142.75± 55.78* (n = 4)	139.17 ± 35.27 (n = 6)	133.83 ± 25.44 (n = 6)
GLU [mmol/L]	16.99 ± 1.82 (n = 6)		14.50 ± 4.85 (n = 6)	13.90 ± 1.08 * (n = 6)	15.40 ± 3.61 * (n = 4)	14.79 ± 2.97 (n = 6)	16.40 ± 5.15 (n = 6)
UREA [mmol/L]	5.97 ± 0.67 (n = 6)		6.17 ± 0.63 (n = 6)	5.40± 0.67 (n = 6)	5.49± 0.52 (n = 5)	6.25 ± 0.46 (n = 6)	6.11 ± 1.50 (n = 5)
CREA [µmol/L]	27.63 ± 9.05 (n = 6)		25.14 ± 5.41 (n = 6)	24.50 ± 3.0 (n = 5)	27.24 ± 5.75 (n = 5)	24.10 ± 6.07 (n = 6)	25.65 ± 12.29 (n = 5)
CHOL [mmol/L]	0.62 ± 0.11 (n = 6)		0.81 ± 0.11 * (n = 6)	0.90 ± 0.16 * (n = 5)	0.82 ± 0.09 * (n = 5)	0.60 ± 0.16 (n = 6)	0.74 ± 0.08 (n = 6)
TP [g/L]	43.63 ± 3.28 (n = 6)		55.08 ± 1.66 * (n = 6)	51.62 ±1.91 * (n = 6)	48.30 ± 1.89 * (n = 5)	56.82 ± 2.76 (n = 6)	58.23 ± 4.77 (n = 6)
Na [mmol/L]	134.83 ± 3.54 (n = 6)		139.33 ± 1.37 * (n = 6)	140.80 ±1.48 * (n = 5)	139.00 ± 1.73 * (n = 5)	140.67 ± 1.97 (n = 6)	141.50 ± 3.39 (n = 6)
K [mmol/L]	4.84 ± 0.53 (n = 5)		4.50 ± 0.28 (n = 6)	4.24 ± 0.18 (n = 5)	4.75 ± 0.29 (n = 3)	4.67 ± 0.51 (n = 6)	5.74 ± 2.55 (n = 6)
ALBB [g/L]	32.12 ± 2.02 (n = 6)		33.80 ± 1.10 (n = 6)	33.22 ± 2.19 (n = 5)	32.22 ±1.93 (n = 5)	32.02 ± 1.27 (n = 6)	32.48 ± 2.14 (n = 5)
TBA [µmol/L]	10.84 ± 3.31 (n = 5)		30.27 ± 21.24 (n = 6)	16.19 ± 2.52 (n = 5)	13.46 ± 6.44 (n = 5)	25.03 ± 12.03 (n = 6)	14.84 ± 6.39 (n = 6)
Females
ASAT [IU/L]	27.87 ± 7.27 (n = 6)	32.57 ± 9.37 (n = 6)		26.58 ± 5.07 (n = 6)	31.28 ± 8.59 (n = 6)	-	-
ALAT [IU/L]	22.78 ± 6.29 (n = 6)	22.93 ± 4.85 (n = 6)		20.48 ± 5.67 (n = 6)	27.43 ± 12.30 (n = 6)	-	-
ALP [IU/L]	201.17 ± 120.65 (n = 6)	194.83 ± 82.27 (n = 6)		164.67 ± 71.03 (n = 6)	157.17 ± 71.74 (n = 6)	-	-
GLU [mmol/L]	7.57 ± 1.40 (n = 6)	7.66 ± 1.96 (n = 6)		7.68 ± 1.70 (n = 6)	7.48 ± 1.25 (n = 6)	-	-
UREA [mmol/L]	11.19 ± 1.28 (n = 6)	12.07 ± 2.42 (n = 6)		10.73 ± 1.47 (n = 6)	11.39 ± 0.91 (n = 6)	-	-
CREA [µmol/L]	29.58 ± 4.41 (n = 5)	24.80 ± 9.02 (n = 5)		20.33 ± 4.12 (n = 5)	27.07 ± 1.99 (n = 5)	-	-
CHOL [mmol/L]	0.73 ± 0.15 (n = 6)	0.83 ± 0.07 (n = 6)		0.81 ± 0.23 (n = 6)	0.90 ± 0.23 (n = 6)	-	-
TP [g/L]	56.00 ± 3.66 (n = 6)	58.00 ± 4.54 (n = 6)		58.05 ± 4.91 (n = 6)	56.70 ± 3.64 (n = 6)	-	-
Na [mmol/L]	144.83 ± 3.60 (n = 6)	150.67 ± 6.02 (n = 6)		148.00 ± 4.77 (n = 6)	149.17 ± 6.68 (n = 6)	-	-
K [mmol/L]	4.83 ± 0.34 (n = 6)	5.46 ± 0.85 (n = 6)		4.99 ± 0.30 (n = 6)	5.17 ± 0.35 (n = 6)	-	-
ALBB [g/L]	37.77 ± 2.81 (n = 6)	38.13 ± 7.21 (n = 6)		34.50 ± 7.56 (n = 6)	38.25 ± 2.51 (n = 6)	-	-
TBA [µmol/L]	8.71 ± 8.73 (n = 6)	8.70 ± 6.31 (n = 6)		12.02 ± 7.11 (n = 6)	14.59 ± 9.78 (n = 6)	-	-

Table 4. Terminal body weight and liver weight [g]
Observation Parameter	At the treatment termination (one day after last treatment)				After 14 days of recovery
	Control	100 mg/kg	250 mg/kg	750 mg/kg	Control	750 mg/kg
Males
Terminal Body weight [g]	357 ± 18 (n = 6)	366 ± 25 (n = 12)	360 ± 31 (n = 12)	357 ± 19 (n = 6)	387 ± 40 (n = 6)	371 ± 23  (n = 6)
Liver weight [g]	11.80 ± 0.78 (n = 6)	13.12 ± 1.62 (n = 6)	14.43 ± 2.44 * (n = 6)	16.15± 1.60* (n = 6)	13.17 ± 1.18 (n = 6)	14.46 ± 1.59 (n = 6)
Females
Terminal Body weight [g]	242 ± 11 (n = 10)	259 ± 20 (n = 12)	248 ± 15 (n = 12)	250 ± 13 (n = 11)	-	-
Liver weight [g]	9.24 ± 0.74 (n = 6)	11.17 ± 1.40 * (n = 6)	11.56 ± 0.96 * (n = 6)	13.31 ± 1.22 * (n = 6)	-	-

Conclusions:

The repeated dose toxicity of the registration substance was investigated according to the OECD Guideline 422. The registration substance induced hemolysis at 750 mg/kg bw at 750 mg/kg bw and liver enlargement in dose-dependent manner in all treated animals. These findings were reversible and there was no indication of functional impairment of blood system and liver. The NOAEL of 750 mg/kg bw was obtained.
Based on the findings obtained in this study, no classification is assigned to the registration substance.

Executive summary:

The repeated dose toxicity of the registration substance was investigated according to the OECD Gudieline 422. Twelve males and females each were treated at doses of 0, 100, 250 and 750 mg/kg bw via gavage. The study included the investigation of the recovery effect in males.Six rats of control and 750 mg/kg bw groups were allowed to recover for 14 days.

At 750 mg/kg bw, the hematology values were altered in males. Together with histopathological findings of extramedullary hemopoiesis and golden-brown pigments in spleens, these findings were considered as indication of hemolysis. Due to the limited degree of the hematology changes and due to the observed reversibility, no adversity was assigned.

Further, liver enlargement was found in dose-dependent manner both for males and females, which were accompanied with slightly changed values of clinical biochemistry and histopathological findings of glycogen storage. As there was no indication of impairment of liver funtion and as comparable values for liver weight were obtained from the animals of the recovery group, the liver enlargement was interpreted as of adaptive nature.

The NOAEL of 750 mg/kg bw was obtained for both males and females.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

750 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The repeated dose toxicity data comprise one subacute toxicity study (OECD 422) on the registrations substance and diverse studies on structurally related substances. The quality of whole database is considered to be sufficiently robust for a reliable hazard assessment on the registration substance.

Additional information

For the assessment of the chronic toxicity of the registration substance, the registration substance was grouped with structurally related chemicals. Following aspects will be discussed in the given order:

a) Justification for the applied read-across approach: basis for the grouping of chemicals

           a1) Group members and the structural similarity

           a2) Mode of action

           a3) Comparable toxicity profiles after prolonged exposure

           a4) Conclusion

b) The chronic toxicity of the registration substance based on grouping of chemicals

           b1) Findings obtained in studies with DEGDBE, DEGBE, and EGBE

           b2) Expected kinetic profile of the registration substance

           b3) Expected toxicity after prolonged exposure based on the proposed read-across approach.

           b4) Expected toxicity for humans based on the EU assessment on EGBE and DEGBE

           b5) Conclusion

 

 

 

a) Justification for the applied read-across approach: basis for the grouping of chemicals

 

The applied read-across approach is based on grouping of chemicals, in which the same mode of action can be presumed.

 

a1) Group members and the structural similarity

 

The similarity in their structure is given by the presence of ”butyl-O-CH₂CH₂-O-“ at terminal position.

Grouping of chemicals for the hazard assessment of the registration substance
Chemicals in read-across approach	Chemical name/ CAS	Structure
Read-across supporting substances	Ethylene glycol butyl ether (EGBE)* CAS 111-76-2	Butyl-O-CH2CH2-OH
	Diethylene glycol butyl ether (DEGBE)* CAS 112-34-5	Butyl-O-(CH2CH2-O)2-H
	Diethylene glycol dibutyl ether (DEGDBE)** CAS 112-73-2	Butyl-O- (CH2CH2-O)2-Butyl
Registration substance/ target chemical	Polyethylene glycol dibutyl ether (PolyEGDBE)*** CAS 31885-97-9	Butyl-O- (CH2CH2-O-)n-Butyl n = 2,3,4

* EGBE and DEGBE are extensively investigated substances and reviews on their toxicity profiles are available in public domain (i.e. EU Risk Assessment Report, 2-Butoxyethanol (EGBE), CAS 111 -76 -2, 2008; Opinion on Diethylene Glycol Monobutyl Ether (DEGBE), SCCP/1043/06, 2006). ** Clariant substance; relevant data provided in corresponding endpoint study record. *** registration substance; target chemical in the read-across.

 

a2) Mode of action

 The proposed grouping is justified by the common mode of action, namely systemic exposure to 2-butoxyacetic acid (2-BAA) and/or butoxyethoxyacetic acid (BEAA):

- EGBE: 2-BAA is the major urinary metabolite (summarized in EU risk assessment, 2008)

- DEGBE: BEAA is the major urinary metabolite (Deisinger et al. 1989)

- DEGDBE: in 28-day study (Clariant own data; details provided in corresponding endpoint study record), the urinary 2-BAA determination was incorporated; 750 mg/kg bw external dose level corresponded to 1400 mg/L 2-BAA in urine.

- PolyEGDBE: no experimental data on metabolite is available; BEAA and/or 2-BAA as metabolite can be reasonably assumed due to the observed RBC reduction and liver enlargement in the OECD 422 study (Clariant own data; provided in corresponding endpoint study record).

 

a3) Comparable toxicity profiles after prolonged exposure

The proposed grouping is justified by the comparable toxicity profiles, which reflects the toxicity action of 2-BAA and/or BEAA. Both metabolites are known to induce hemolysis (Udden 2002; Udden 2005).

- EGBE: hemolytic action demonstrated in acute and repeated dose toxicity studies (summarized in EU risk assessment, 2008; selected studies provided in corresponding study record)

- DEGBE: i.e. in 2 and 13 week oral toxicity studies, RBC reduction was evident. (Johnson et al. 2005; provided in corresponding study record)

- DEGDBE: in 28-day study, RBC reduction and hematuria was evident (Clariant own data; provided in corresponding study record)

- PolyEGDBE: in OECD 422, RBC reduction was evident (Clariant own data; provided in corresponding study record)

 

a4) Conclusion: Based on a2) and a3), the introduced grouping is justified, which in turn justifies the use of the toxicity data of each group members for the hazard assessment of the registration substance.

 

 

b) The chronic toxicity of the registration substance based on grouping of chemicals

 

The toxicity data on the registration substance itself is limited to one subacute toxicity study (OECD 422). The treatment related effects found at 750 mg/kg bw were indicative of hemolysis. Because there was no indication of impairment of blood system and the reversibility after 14 days of recovery could be demonstrated, the dose level of 750 mg/kg bw was assigned as the NOAEL. In addition, the liver enlargement was found for all treated animals in dose-dependent manner. However, the liver effect was considered as signs of the metabolic adaptation and therefore, less relevant for the chronic toxicity.

 

b1) Findings obtained in studies with DEGDBE, DEGBE and EGBE

In all studies presented in this dossier, the red blood cell was identified as the target organ and the basis for the NOAELs was the adversity of hemolysis. By comparing the obtained NOAELs, a potency order of EGBE > DEGDBE > DEGBE ≈ registration substance can be obtained.

 

Table: Overview of the repeated dose toxicity studies of the grouped chemicals and NOAELs
Group member	Study type	NOAEL /LOAEL
Registration substance PolyEGDBE	OECD 422	NOAEL of 750 mg/kg bw
DEGBE	13-week toxicity study	NOAEL of 250 mg/kg bw
DEGDBE	28-day toxicity study	NOAEL of 100 mg/kg bw
EGBE	14 week inhalation	LOAEC of 31 ppm (150 mg/m^3)
	104 week inhalation	LOAEC of 31 ppm (150 mg/m^3)

* The mentioned studies are provided in corresponding study record

 

b2) Expected kinetic profile of the registration substance

The 28 -day study with DEGDBE comprised collection of urine samples after 8 and 28 days exposure and after recovery of 1 and 14 days (the endpoint study record provided under 7.1. in IUCLID). These samples were analyzed for the metabolite 2-butoxyacetic acid (2-BAA), which was found as the ultimate toxicant for the hemolysis (Udden 2002; Udden 2005). Comparable values were obtained in the samples of 8 and 28-day treatment and very low levels in the samples of recovery animals. These values are demonstrating that the steady state of the systemic exposure was already established within 8 days. Also the obtained very low levels in the recovery animals, especially already after one day of recovery, are indicating efficient elimination capacity. DEGDBE is a non-bioaccumulating substance, and likewise no significant bioaccumulating property can be assigned for the registration substance.

 

b3) Expected toxicity after prolonged exposure for the registration substance

Considering that:

-         the overall data on the group members include up to the studies of 2 years

-         the registration substance is of the lowest potency among the group members

-         and no bioaccumulation can be soundly derived,

it is reasonable to consider the observed subacute toxicity of the registration substance as representative for subchronic/chronic toxicity. A low chronic toxicity can be derived.

 

b4) Expected toxicity for humans based on the EU assessment on EGBE and DEGBE

According to the official EU documents (EU Risk Assessment 2 -Butoxyethanol Cas 111 -76 -2, ECB, 2008; Opinion on Diethylene Glycol Monobutyl Ether (DEGBE) SCCP/1043/06, 2006) EGBE or DEGDBE are agents causing hemolysis in rats and mice. Humans are likely to be more resistent, so that toxic effects in such a degree found in animal studies cannot be expected. Based on the grouping of chemicals, it is justified to apply the same consideration also for the registration substance. The chronic toxicity of the registration substance for humans is expected to be low.

 

b5) Conclusion: Based on the provided arguments b1)-b4) the registration substance is considered as of low chronic toxicity.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Scientifically well-performed; Guideline study; GLP study; study on the registration substance

Justification for classification or non-classification

The registration substance was investigated for its repeated dose toxicity according to the OECD Guideline 422. Indications of hemolysis and metabolic adaptation were found. No significant toxic effect was found up to the dose of 750 mg/kg bw.

For the assessment of the chronic toxicity of the registration substance, the read-across approach was introduced. Based on the knowledge on the mode of action/kinetic profile/chronic toxicity of the read-across supporting substances, the hazard profile of the registrations substance after prolonged exposure was predicted to be comparable to that found in the OECD 422 study. No classification is assigned.