Registration Dossier

Administrative data

short-term repeated dose toxicity: oral
Combined 28-Day Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The substance can be produced through different process routes, yielding solutions in water or organic solvent (e.g. methanol or ethylene glycol).
It was initially unclear how to register the substance (mono-constituent or multi-constituent substance) and which manufactured substance to test to fulfil the REACH data requirements. After consultation with the ECHA helpdesk, the test program was started with the manufactured substance of the Lead registrant (solvent: methanol) in which the highest amount of solvent could be removed without causing degradation of the substance. This resulted in the selection of a solution of 56-57% BTMAOH in methanol as test substance.
During the course of the test program, and in order to aid meaningful risk assessment, after consultation with ECHA and upon ECHA's recommendation, it was considered to be more appropriate to test the water-based manufactured substance. As a consequence, some testing was performed with a BTMAOH solution in methanol, and some testing was performed with a BTMAOH solution in water.

The current entry reflects a test performed with a water-based test solution.

Data source

Reference Type:
study report

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 2016
according to
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)
Version / remarks:
July 2000
Principles of method if other than guideline:
In addition, the design of this study is essentially conform to the following guidelines.
• OECD Guideline 421. Reproduction/Developmental Toxicity Screening Test, July 2016.
• EPA Health Effects Test Guideline OPPTS 870.3550: Reproduction/Developmental Toxicity Screening Test, July 2000.
• Council Regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.
• OECD Guideline 407. Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.
• EPA Health Effects Test Guideline OPPTS 870.3050: Repeated dose 28-day oral toxicity study in rodents, July 2000.
GLP compliance:
Limit test:

Test material

Test material form:
Details on test material:
Appearance: Clear almost colourless liquid
Test item storage: At room temperature
Stable under storage conditions until: 30 November 2020 (expiry date)
Specific details on test material used for the study:
pH: 13 at concentration of 20%

Test animals

other: Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source.
This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Details on test animals and environmental conditions:
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: to be included
- Weight at study initiation: to be included
- Fasting period before study: to be included
- Housing: On arrival and following pre-mating period, animals will be group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). During the mating phase, males and females will be cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, males will be housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females will be individually housed in Macrolon plastic cages (MIII type, height 18 cm). During the lactation phase, pups will be housed with the dam, except during locomotor activity
monitoring of the dams, when the pups will be kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups should not be left without their dam or a bottle filled with warm water for longer than 30-40 minutes. For psychological/environmental enrichment and nesting material, animals were provided with paper.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum throughout the study, except during designated procedures. During motor activity measurements, animals did not have access to food for a maximum of 2 hours.
- Water: Municipal tap water ad libitum. During motor activity measurements, animals did not have access to water for a maximum of 2 hours.
- Acclimation period: at least 5 days

- Temperature (°C): 18-24
- Humidity (%): 40 - 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12


Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
Details on oral exposure:
For dosing of Group 4 animals: The test item was administered as a solution as received. An adequate amount of the test item was dispensed into daily aliquots, which was stored in a controlled temperature area set to maintain 21°C until use.
For dosing of Group 2 and 3 animals: Test item dosing formulations (w/w) was homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared weekly as a solution formulated in daily portions and stored in the refrigerator protected from light. The dosing formulations were removed from the refrigerator and stirred at room temperature for at least 30 minutes before dosing and dosed within 24 hours after removal from the refrigerator.
Test item dosing formulations were kept at room temperature until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during
dosing. Adjustment was made for specific gravity of the vehicle. A factor of 4.92 was used to correct for the purity/composition of the test item.

Dose volume: 0.0717 mL/g
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
For Group 4, the test item was used as received from the Sponsor; therefore, samples for dose formulation analysis for Group 4 were not collected by the Test Facility.
Samples for Analysis: Duplicate middle samples for Groups 1 (concentration analysis only) and duplicate top, middle, and bottom samples for Groups 2 and 3 (concentration and
homogeneity analysis).
Sample Volume: Approximately 500 mg accurately weighed.

Details on analytical veryfication: to be included
Acceptance Criteria: For concentration: mean sample concentration results within or equal to ±10% for solutions of target concentration; For homogeneity: coefficient of variation (CV) of concentrations of ≤ 10% for each group.
Duration of treatment / exposure:
Males minum of 28 days, females 14 days prior to mating, the variable time to conception, the duration of pregnancy and at least 13 days after delivery.
Frequency of treatment:
Once daily, 7 d/w
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Group 1
Dose / conc.:
1.5 mg/kg bw/day (actual dose received)
Group 2
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Group 3
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Group 4
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were based on results of a 28-Day dose range finding study with oral gavage of the test item.

Results and discussion

Effect levels

Key result
Remarks on result:
not measured/tested
Study is still ongoing.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Results dose range finder:

A dose range finder was conducted previously to select dose levels for the main study and to determine the peak effect of occurrence of clinical signs after dosing. No testing guidelines were applicable as this dose range finder was intended for dose level selection purposes only.

A dose of 100 mg/kg was not tolerated by the animals as severe clinical signs, reduced food consumption and mild to severe body weight loss were observed. All animals treated with 100 mg/kg died before scheduled necropsy on Day 2, 5 and 6 of treatment. At dose level 25 mg/kg, some clinical symptoms, reduced food consumption and slight body weight loss were observed during the 14 Days of treatment. One animal at 25 mg/kg died at Day 8 of treatment. A dose of 15 mg/kg was well tolerated for 28 days and therefore a maximum dose level of 15 mg/kg was selected for the main study. Because of the high pH of the test item and its corrosive properties, the dose volume for the main study was set at 0.072 mL/kg, based on the dose volume used for the 15 mg/kg Group of the dose range finder. In the dose range finder, clinical signs were generally observed directly after dosing.

Applicant's summary and conclusion